Per-well illumination and optical stimulation arrangements for advanced microplate, microarray, and microtiter technologies

ABSTRACT

Arrangements for per-well illumination and optical stimulation arrangements for microplate, microtiter, and microarray technologies are presented. In example implementations, each individual well within in a conventional or specialized microplate can be fully or partially isolated with capping or other arrangements which can include conduits for controlled introduction, removal, and/or exchange of fluids and/or gases. Conduit networks can include small controllable valves that operate under software control, and micro-scale pumps can also be included. Conduit interconnections can include one or more of controllable-valve distribution buses, next-neighbor interconnections, and other active or passive interconnection topologies. Cap arrangements can include or provide one or more sensors of various types, including but not limited to selective gas sensors, chemical sensors, temperature sensors, pH sensors, biosensors, immunosensors, molecular-imprint sensors, optical sensors, fluorescence sensors, bioFETS, etc. Incubator interfacing and imaging are also described. The invention can be used for living cell culture or other applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of pending U.S. patent applicationSer. No. 13/844,621, filed Mar. 15, 2013, the contents of which areincorporated by reference.

COPYRIGHT & TRADEMARK NOTICES

A portion of the disclosure of this patent document may containmaterial, which is subject to copyright protection. Certain marksreferenced herein may be common law or registered trademarks of theapplicant, the assignee or third parties affiliated or unaffiliated withthe applicant or the assignee. Use of these marks is for providing anenabling disclosure by way of example and shall not be construed toexclusively limit the scope of the disclosed subject matter to materialassociated with such marks.

BACKGROUND OF THE DISCLOSURE Field

Embodiments of the application pertain generally to cell incubators,microarrays, microfluidic systems, and miniature biochemical andchemical detectors, and more specifically to microprocessor-controlledmicrofluidic platform technologies comprising such miniature biochemicaland chemical detectors configured as instrumented cell incubators forproviding a plurality of simultaneous distinct controlledmicro-environments for living cell cultures.

GENERAL BACKGROUND

Culturing cells, such as tissue cells, cancer cells, bacteria, yeasts,molds, plankton, infectious protozoa, etc. as well as cell-relatedmaterials such as infectious viruses and prions in the laboratorytypically require managed, and often precisely managed, environmentalcontrol. In the laboratory, cell incubators are the most common andessential equipment for nurturing and maintaining living cells.

Cell incubators typically provide several environment-providingfunctions that often must meet or operate within numerous rigidspecifications relating to temperature, humidification, gaseousenvironment, sterilization, and specimen safety. Additionally theseconditions are not uniform across cell types. For example, culturingmammalian cells and bacteria require the temperature at 37° C., 5%carbon dioxide, and 95% humidity and sterilized condition. However,other cell types, for instance the budding yeast Saccharomycescerevisiae grows at a temperature at 30° C.

Contemporary cell incubator technologies can provide computer controlledthermal regulation, humidified control, control of gas levels such asCO₂, O₂, and N₂, and illumination of cell cultures contained in open (orin some cases closed) dishes and welled microplates. In addition, manycontemporary cell incubators can be programmed to control environmentalfactors through a sequence or cycle of distinct temperatures, humiditylevels, etc.

In vitro study can involve study of biochemical process orpharmacodynamical substances provided to cultured cells. Examplesinclude adding pharmaceuticals and changing aspects of the culturemedium. In order to enact these, the sample must typically be removedand translocated to outside the incubator, thus discontinuing ordisrupting the controlled cell nurturing condition. In contrast to invitro study, in vivo study provides a consistent biological environmentfor observing biological responses from the effects of medicaltreatment. Uninterrupted controlling of the conditional environmentwhile studying cellular responses can minimize the introduction ofcorrupting processes and far more precisely mimic the actual biologicalenvironment of the body of a higher organism or other natural cellularenvironment.

In addition, the required and optimal conditions of the biologicalenvironment for various cell types are often different, especially inregards to the proportion of ambient gases such as carbon dioxide (CO₂),oxygen (O₂) and nitrogen (N₂). For example, cells within a solid tumormalignancy are in hypoxia (low oxygen), yet normal cell is in normoxia(normal oxygen). Additionally, hypoxia conditions play an important rolein gene expression related to cellular signal transduction. Therefore,to study the cellular responses at the desired cell cultureconditions—hypoxia and normoxia, control of the proportion of gasesduring cell incubating is considered.

To study effects of substance concentrations on the biologicalresponses, monitoring levels of the interested substances are concerned.For example, different concentrations of nitric oxide (NO) are relevantto cell signaling and cell apoptosis, measuring concentration of nitricoxide at steady states is required during cell culture. In some cases,substances of interest are produced naturally by biological process,while in other types of experiments, substances of interest areintroduced artificially.

Further, it is noted for example that due to its highly reactive nature,artificially provided NO typically must be introduced very locally to NOaffectation/consumption regions by controlled introduction of NO-donorcompounds. Other types of substance concentration experiments canrequire highly localized substance measurement (for example by means ofsubstance-responsive fluorescent markers) and/or highly localizedsubstance introduction (for example by means of substance-generativedonors).

To study the pharmacological and biochemical effects via in vitro study,observing cell responses from within incubation—i.e., without removaland interruption of the period of environmental control, would beexpected to provide higher accuracy and diminish corruptionopportunities. However, current methods for determining the effects ofthe substances on the cells involve waiting until cells are stable inthe cell culture medium and environment and subsequently performing anoperation outside the environmental control provided by the incubator.Even though efforts can be taken to minimize analysis time outside theincubator can reduce corruptive effects, often cells are still verysensitive to changes of environment. In addition, other aspects of cellresponse study can be affected and made more complicated. Therefore,processes such as treating the cells with substances and analyzing thecell responses against monitored desirable conditions, and operations ofprocesses within the control condition in the incubator have become aformidable challenge for in vitro studies.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other aspects, and features of the present applicationwill become more apparent upon consideration of the followingdescription of embodiments taken in conjunction with the accompanyingdrawing figures, wherein:

FIG. 1 depicts a representation of an example microplate comprising cellincubation wells.

FIG. 2 depicts example fluidics arrangements included within amicroplate.

FIG. 3 depicts an incubation system according to some embodiments.

FIG. 4a depicts an example arrangement suitable for providing a commongas environment to all cell cultures in an incubator system.

FIG. 4b depicts an example arrangement suitable for providing a commongas environment to all cell cultures in an incubator system.

FIG. 5 depicts an example arrangement wherein a microplate includingfluidics arrangements.

FIG. 6a depicts an example of a relatively complex microscopic imagingcamera arrangement.

FIG. 6b depicts an example of a simplified microscopic imaging cameraarrangement.

FIGS. 7a and 7b depict an arrangement in which a microplate is insertedinto one of a plurality of compartments within the incubator.

FIGS. 8a-8c depict an arrangement in which a microplate is inserted intoa microplate enclosure that in turn is configured to be inserted intoone of a plurality of compartments within the incubator.

FIGS. 9a-c depict an arrangement in which a microplate is fitted with anenvironment-localizing microplate cap.

FIG. 10a depicts a representation of an example cross-section of anindividual well of a microplate (such as that depicted in FIG. 1) andimmediately surrounding microplate material.

FIG. 10b depicts a representation of an example separate self-containedcap and associated fluidic/gas-tight seal that can be placed against theopen portion of the microplate well depicted in FIG. 10 a.

FIG. 10c depicts a variation on the arrangement depicted in FIG. 10bwherein the cap is not a separate self-contained structure but insteadrendered as a well or cavity of a larger piece of structural material.

FIG. 11a depicts a variation on the arrangement depicted in FIG. 4bwherein the centralized gas mixture operation is replaced with amultichannel gas distribution bus and controllable amounts of selectedgas components are delivered individually to each well and cap chamber.

FIG. 11b depicts a variation on the arrangement depicted in FIG. 11awherein separate post-distribution gas purification is provided for atleast some of the chambers created by well and cap pairs.

FIG. 12 depicts a system for providing a plurality of separatelycontrolled environments, separately controlled reagent introduction, andseparate monitoring, each for an associated cell culture or group ofcell cultures according to certain embodiments.

FIG. 13 depicts an example arrangement wherein cell culture media fluidflows from a fluid source to individual cell culture wells and fromthere to fluidic waste handling, and wherein reagents such as hormones,signaling donors, stains, etc. can be introduced into cell culture wellsby fluids.

FIG. 14a and FIG. 14b depict example arrangements for providing gentlefluid flow over a ramped circular flow arrangement around a rim of awell.

FIG. 15 depicts an arrangement wherein the rate and contents of flowdelivery is individualized by connecting arrangements such as those ofFIG. 14a or FIG. 14b to an instance or adaptation of controllablemicrofluidic bus technologies.

FIG. 16a depicts an example arrangement wherein fluids can be introducedfrom below the cell wells.

FIG. 16b depicts an example arrangement wherein fluids can betransported in and out of individual cell culture wells bysurface-exposed trays.

FIG. 16c depicts an example arrangement involving a combination of theapproaches of FIGS. 16a and 16 b.

FIG. 17a depicts simplified unified view of the basis of biosensingtechnologies.

FIG. 17b provides a view of the diversity of biosensor technologies andapproaches suitable or adaptable for full microsystem implementation.

FIG. 18 depicts a representation of new and adapted individual componenttechnologies provided for by various embodiments.

FIG. 19 depicts an adaptive framework provided and performed byembodiments of the present application so as to create a flexiblemultiple-purpose platform technology.

FIG. 20 depicts an overall overview of the software, signal inputhardware, signal processing hardware, and software-control hardwareprovided for or implemented in various embodiments of the presentapplication.

FIG. 21 depicts an example abstract representation of a removablereplaceable media element.

FIG. 22a depicts an embodiment of a system comprising a removablereplaceable media element in communication with a base unit.

FIG. 22b depicts a simple high-level combined signal-flow andfluidic-flow representation of a system comprising a removablereplaceable media element in communication with a base unit.

FIGS. 23a-23c depict possible user and interface implementationsaccording to some embodiments of the present application.

FIGS. 24a-24d depict example representations of the removablereplaceable media element according to some embodiments.

FIG. 25 depicts an example representation of the offset bottom view of a“cap” that meets and covers each site on the removable replaceable mediaelement with a fluid-tight seal.

FIGS. 26a-26b depict representations of example functional printedmethods that can be used to print the sensors on the removablereplaceable medium.

FIG. 27a depicts a cap interfacing with a site on the removablereplaceable media element according to some embodiments.

FIG. 27b depicts a cap interfacing with a site on the removablereplaceable media element that comprises a printed sensor according tosome embodiments.

FIG. 27c depicts a cap interfacing with a site on the removablereplaceable media element that comprises a printed deposition accordingto some embodiments.

FIG. 28 depicts a cap with a fluidic port accepting solvent in and afluidic port carrying solvent and reagent outward according to someembodiments.

FIG. 29 depicts an example arrangement wherein caps interconnected withfluidics arrangements interface to associated sites on a portion of theremovable replaceable media element according to some embodiments.

FIGS. 30a-30c depict examples of how optical ROM printed on theremovable replaceable media can be read by the base system according tosome embodiments.

FIG. 31 depicts an example adaptation of the example architecturalarrangement provided in FIG. 22b wherein a removable replaceableinterface module is provided interfaces to the microfluidics andcomputing infrastructure.

FIG. 32 depicts an embodiment in which at least the fluidicsarrangements are comprised in an interfacing module.

FIG. 33 depicts an embodiment in which the interfacing module can beconfigured to be inserted into either the base system or attached to theremovable replaceable media element in either a fixed or replaceablearrangement.

FIG. 34 depicts a representation of applications software that caninclude, for example, configuration data, assignment data, data used byalgorithms, test algorithms, analysis algorithms, etc.

FIG. 35 depicts a representation of an example user experience scenariousing an example implementation of the technology.

FIG. 36 depicts a representation of the strategy of an exampleexperiment facilitated by various embodiments.

FIG. 37 depicts a flow chart of an example experiment leveraging variousaspects of various embodiments.

FIG. 38 depicts a representation of how measurement data obtained fromparallel hypoxia and normoxia conditions are compared.

FIG. 39 depicts a representation of how NO donor material profiles canbe studied.

FIG. 40 depicts a representation of experimental processes for studyingeffect of NO on cell survival.

FIG. 41 depicts the principle of operation of an example NO quantitativekit.

FIG. 42a depicts a summarization of the relationship between NO exposureconcentration and duration (control variables in the experiment) andcell survival time.

FIG. 42b illustrates a method for determining cell response to normoxicconditions.

FIG. 42c illustrates a method for determining cell response to hypoxicconditions.

FIG. 42d illustrates a method for determining cell response to hypoxicconditions.

FIG. 43a and FIG. 43b depict representative tables useful in the studyof the releasing kinetics in hypoxic and normal conditions of various NOdonors.

FIG. 44 depicts an example representation of the effect of oxygenconcentration (pO₂) on O₂—, NO and ONOO— production.

FIG. 45 demonstrates the results of a simulation of Phenobarbital plasmaconcentration during repeated oral administration for the applicationschemes indicated. Based on Vd=38 L, CL=0.26 L/h (t1/2=100 h), F=1.

FIG. 46 demonstrates steady state NO concentration showing NO generationfrom DETA/NO in the absence and presence of anaerobically grown N.gonorrhoeae.

DETAILED DESCRIPTION

In the following description, reference is made to the accompanyingdrawing figures which form a part hereof, and which show by way ofillustration specific embodiments of the present application. It is tobe understood by those of ordinary skill in this technological fieldthat other embodiments may be utilized, and structural, electrical, aswell as procedural changes may be made without departing from the scopeof the present application.

In the following description, numerous specific details are set forth toprovide a thorough description of various embodiments. Certainembodiments may be practiced without these specific details or with somevariations in detail. In some instances, certain features are describedin less detail so as not to obscure other aspects. The level of detailassociated with each of the elements or features should not be construedto qualify the novelty or importance of one feature over the others.

Various embodiments provide for various synergistic combinations of thefollowing features:

-   -   Dedicated localized sensors associated with individual wells of        replaceable removable media        -   In replaceable removable media well        -   In associated cap sealing the top of replaceable removable            media well        -   Distributed among well and cap    -   Dedicated localized fluidics associated with individual wells of        replaceable removable media        -   In replaceable removable media well        -   In associated cap sealing the top of replaceable removable            media well        -   Distributed among well and cap    -   Shared gas exchange for a group of individual wells of        replaceable removable media    -   Dedicated localized gas exchange associated with individual        wells of replaceable removable media        -   In replaceable removable media well        -   In associated cap sealing the top of replaceable removable            media well        -   Distributed among well and cap    -   Shared thermal control for a group of individual wells of        replaceable removable media    -   Dedicated localized thermal control associated with individual        wells of replaceable removable media        -   In replaceable removable media well        -   In associated cap sealing the top of replaceable removable            media well        -   Distributed among well and cap    -   Shared imaging for a group of individual wells of replaceable        removable media    -   Dedicated localized imaging associated with individual wells of        replaceable removable media        -   In replaceable removable media well        -   In associated cap sealing the top of replaceable removable            media well        -   Distributed among well and cap.    -   Shared illumination for a group of individual wells of        replaceable removable media (with some embodiments providing a        selectable range or combination of illumination wavelengths in        the visible and/or U.V. range)    -   Dedicated localized illumination associated with individual        wells of replaceable removable media (with some embodiments        providing a selectable range or combination of illumination        wavelengths in the visible and/or U.V. range)        -   In replaceable removable media well        -   In associated cap sealing the top of replaceable removable            media well        -   Distributed among well and cap.

These arrangements provide for various degrees of individuallycontrolled, monitored, and isolated cell incubation chambers byleveraging computer-executed algorithms and computer-interfacinghardware for providing computer monitoring and computer control.

Additionally various embodiments employ several component core, design,and fabrication technologies including one or more of:

-   -   One or more arrays of individual incubator chambers, each        comprising microfluidic and biosensor aspects.    -   Microplates and associated fluidic structures can be fabricated        employing function printing, 3D printing, and/or digital        printing.    -   Printed electronic sensors or sensor components associated with        individual chambers, for example employing one or more of        printed organic semiconducting and printed conducting polymers,    -   Optical sensors or sensor components associated with individual        chambers employing one or more of printed antibodies, printed        optical materials, printed organic semiconducting, printed        conducting polymers,    -   Printed mechanical structures associated with individual        chambers,

FIG. 1 depicts a representation of an example microplate 100 comprisingcell incubation wells 110. Such microplates 100 are commonly used for avariety of purposes and often are used to contain arrays of materialssuch as biological substances and cell cultures. Typically suchmicroplates are passive tray-like structures similar in many ways tokitchen refrigerator ice trays. The wells 110 are spatially isolated forone another and accordingly can be used to contain individual segregatedcell cultures. Further, the cell cultures can be provided withcolorimetric indicators or fluorescent markers whose optical absorptionand emission properties are responsive to biological molecules orconditions.

In some embodiments, microplate 100 can be provided with additionalenhanced features, for example:

-   -   Miniature electrical biosensors (electrochemical, bioFET, etc.)        comprised within wells 110 of the microplate 100;    -   Miniature optical biosensors comprised in whole or in part        within wells 110 of the microplate 100 or otherwise associated        with wells 110 of the microplate 100;    -   Passive (flow passages) fluidics integrated into the microplate        100 and directly connected to wells 110 of the microplate 100,        as discussed further with reference to in FIG. 2;    -   Active (valves, pumps, electro-osmosis, electro-wetting, etc.)        fluidics integrated into the microplate 100, and in some cases        directly connected to wells 110 of the microplate 100.

As to miniature biosensors, a wide range of suitable miniatureelectrical biosensors and miniature optical biosensors are taught inpending U.S. patent application Ser. No. 13/761,142. These can befabricated by functional printing for example as taught in pending U.S.patent application Ser. No. 13/761,142 and as will be explained.

As to fluidics, fluidics arrangements can be included within amicroplate 200, for example as suggested by the arrangement depicted inFIG. 2. Microplate 100 comprising intricate fluidic structures 220 thatcan be fabricated, e.g., by employing function printing, 3D printing,and/or digital printing methods. As illustrated in FIG. 2, fluidicstructures 220 are directly connected to wells 210 of microplate 200.Various approaches to removable controlled fluidics systems and methodsare taught in pending U.S. patent application Ser. No. 13/761,142 andthe co-filed U.S. patent application entitled “Removable FluidicsStructures for Microarray, Microplates, Sensor Arrays, and otherRemovable Media.”

In some embodiments, fluidics arrangements 220 within a microplate 200can be included and configured to provide individual wells 210 in themicroplate 200 a controlled fluidic commonly or individually dispenseddelivery of nutrients. In some embodiments, fluidics arrangements 220within a microplate 200 can be included and configured to provideindividual wells 210 in the microplate 200 a controlled fluidic commonlyor individually dispensed delivery of dissolved gases. In someembodiments, fluidics 220 can also be configured to provide regulatedlocal thermal control, and can be configured to commonly or individuallydispense protective materials to prevent or fight unintended infectionsfrom various phages, pathogens, parasites, and competing intruder celltypes. The fluidics 220 can also be used to commonly or individuallyintroduce materials to be exposed to the cells, for example drugs,pharmaceutical agents, photosensitizers, fluorophore probes/markers,etc. into individual wells 210. Additionally, fluidics 220 in themicroplate 200 can provide controlled fluidic removal of waste materialsso as to support life processes of one or more cells in an adaptedand/or modified cap and site arrangement.

FIG. 3 depicts an incubator system 300 for providing computer controlledthermal regulation, humidity control, and control of gas levels (such asCO₂, O₂, and N₂) for cell cultures contained in an open welledmicroplate, the microplate fitted, for example, with biosensors and/ormicrofluidics.

Incubator system 300 comprises an incubation chamber 310 for receivingone or more microplates and for providing a common atmospheric chamberpresented to open wells of cell culture, a dispenser 330 for dispensingcell culture media/reagent, biosensor 340, a gas input source 350 and acontroller 320 to provide one or more of the following control features:

-   -   For the entire set of wells of a microplate:        -   Gas handling, as discussed further with reference to FIGS.            4A and 4B;        -   Controlled illumination of cells, for example to photoactive            specific materials, for imaging, for colorimetric or            fluoroimetric measurement, photosynthesis, etc.;        -   Programmed control of one or more environmental factors            (temperature, humidity, gas environment, etc.) through a            sequence or cycle of distinct temperatures, humidity levels,            etc.; and        -   Imaging capabilities.

In some embodiments, individual wells within a microplate comprise oneor more of:

-   -   A. Miniature electrical biosensors (electrochemical, bioFET,        etc.) comprised within wells of the microplate;    -   B. Miniature optical biosensors comprised in whole or in part        within wells of the microplate or otherwise associated with        wells of the microplate;    -   C. Passive (flow passages) fluidics integrated into the        microplate;    -   D. Active (valves, pumps, electroosmosis, electrowetting, etc.)        fluidics integrated into the microplate.

In some embodiments, incubator system 300 provides gas handling for theentire group of wells of a microplate. FIG. 4a depicts an examplearrangement suitable for providing a common gas environment to all cellcultures in the incubator.

In this example, gases are fixed mixed 410 under computer control (e.g.,control by controller 320) and then purified 420 (for example operationsinvolving filtration and sterilization by U.V. light). The resultingpurified gas mixture 430 can then be provided to a common atmosphericchamber presented to open wells or dishes of cell culture, such asincubation chamber of FIG. 3. Thus FIG. 4a depicts an examplearrangement suitable for providing a common gas environment to all cellcultures in an incubator wherein gases are first fixed mixed undercomputer control, then purified (for example involve options involvingfiltration and sterilization by U.V. light), and provided to a commonatmospheric chamber presented to open wells or dishes of cell culture,for example in particular to embodiments such as that of earlier FIG. 3.

Alternatively, FIG. 4b depicts an example variation on the arrangementof FIG. 4a wherein each gas is separately purified first prior to mixingbefore a resultant purified gas mixture 440 is provided to a commonatmospheric chamber presented to open wells or dishes of cell culture,such as incubation chamber of FIG. 3.

FIG. 5 depicts an example a microplate 500 including wells 510 withfluidics arrangements 520. Also illustrated is a microplate cap 505,e.g., which may be used to compartmentalize a microplate from othermicroplates within incubator system. In some embodiments, microplate 500operates within a common gas, temperature, humidity, illumination, andimaging environment provided by an incubator, such as incubator system300. Incubator system (e.g., incubator system 300) provides gas input540, cell medium 550 and humidity 560 and controls a thermal input 570to microplate 500. Microplate 500 may receive plate-specific gas andmedia flows and temperature regulation. A dispenser (e.g., dispenser330) dispenses cell culture media/reagent 580.

In some embodiments, the arrangement depicted in FIG. 5 can beconfigured to provide microscopic imaging, for example, by employing amovable microscopic imaging camera. In an embodiment, wells fitted withsensors and/or fluidics in a microplate can be configured to have atransparent region in the well base that can be illuminated from below.In some embodiments, the entire collection of wells is simultaneouslyilluminated. In other embodiments, each well (or small group of wells)is individually illuminated. In some embodiments, the imaging lens orlens system together with imaging camera can be moved in order to imageone or more selected wells. In other embodiments, the entire collectionof wells are simultaneously illuminated and a plurality of imaging lensor lens systems together with imaging camera are arranged in fixedlocations dedicated to the microscopic imaging of one or more selectedwells.

FIGS. 6A and 6B illustrate examples of microscopic imaging cameraarrangement 600 and 650. In both arrangements, a well (610, 660respectively) having a corresponding transparent bottom (615, 665respectively) is illuminated from below using light source (620, 655respectively). Arrangement 650 utilizes a simple imaging lens 680 sothat an image is sensed by imaging sensor 690. Arrangement 600 utilizesa more complex lens system 630 and condenser 640, so that an image issensed by imaging sensor 645. FIGS. 6A and 6B also illustrate that wells(610, 660 respectively) may be fitted with sensors (shown) and fluidics(not shown).

Individualized Environments

The arrangements and example embodiments described thus far providevarious combinations of controlled fluidic inflow/outflow individualizedto each well and individually monitoring of each well. However, thearrangements and example embodiments described thus far have a remaininglimitation in that they provide the same gas, temperature, humidity,illumination, and imaging for all wells in the incubator. Additionalembodiments provide several approaches to overcome this limitation, forexample:

-   -   1. Supporting a plurality of microplates in the incubator and        providing a plurality of separate individualized environments to        subsets of the plurality of microplates—in the extreme an        embodiment can be configured to provide separate individualized        environment to each microplate in the incubator.    -   2. Providing a plurality of separate individualized environments        to subsets of wells of the microplates—in the extreme an        embodiment can be configured to provide separate individualized        environment to each well in a microplate.    -   3. Providing a combination of these approaches.

As example of approach 1 (and its role in approach 3), variousembodiments can be configured to provide a plurality of separatelyindividualized incubator environments, each accepting one or moremicroplates. As an example, not to be viewed as limited, FIG. 7a depictsan arrangement wherein a microplate 700 is inserted into one 710 of aplurality of compartments within an incubator, resulting in the nestedconfiguration depicted in FIG. 7b . The microplate 700 enclosure withinthe incubator compartment 710 provides individualized plate-specific gasand media flows, temperature regulation, illumination, imaging, etc.

As another example of approach 1 (and its role in approach 3), not to beviewed as limited, FIG. 8a depicts an arrangement wherein a microplate800 is inserted into a microplate enclosure 810 which in turn isconfigured to be inserted into one 820 of a plurality of compartmentswithin an incubator as shown in FIG. 8b , resulting in the nestedconfiguration depicted in FIG. 8c . The microplate enclosure within theincubator compartment provides individualized plate-specific gas andmedia flows, temperature regulation, illumination, imaging, etc.

As a further example of approach 1 (and its role in approach 3), not tobe viewed as limited, FIG. 9a depicts an arrangement wherein amicroplate 900 is fitted with an environment-localizing microplate cap910. The microplate 900 and fitted microplate cap 910 are configured tobe inserted into one 930 of a plurality of compartments within theincubator as shown in FIG. 9b , resulting in the nested configurationdepicted in FIG. 9c . The arrangement within the incubator compartmentprovides individualized plate-specific gas and media flows, temperatureregulation, illumination, imaging, etc.

Regarding gas handling, aspects of the arrangements such as thatdepicted in FIG. 4b and FIG. 4a can be replicated for each such instanceof FIG. 7b , FIG. 8c , FIG. 9c , or related nested arrangements.Temperature and humidity control, illumination, imaging, and otherarrangements can be similarly replicated. Alternatively, with regards togas handling, a multichannel gas distribution bus can be used (insteadof replicating aspects of the arrangements such as that depicted in FIG.4b and FIG. 4a ) wherein controllable amounts of selected gas componentsare delivered individually to each well and cap chamber. Such amultichannel gas distribution bus can be realized, for example, as (orusing the same systems and methods as) a controllable multichannelmicrofluidic chemical bus such as that taught in pending U.S. Pat. No.8,032,258 and pending U.S. patent application Ser. Nos. 13/251,286 and13/251,288.

As to approach 2 (and its role in approach 3), the cap and sitearrangement taught in pending U.S. patent application Ser. No.13/761,142 can be adapted and/or modified to support living cells,either individually or in culture. For example, in the context ofpending U.S. patent application Ser. No. 13/761,142, the microplatedepicted in FIG. 1, either in standard commercially available passive(“ice tray”) form, fitted with biosensors, fitted with fluidics, etc.can be regarded as the “removable replaceable media element” taughttherein.

-------CAP and WELL

FIGS. 10a-10c depicts representation of an example cross-section of anindividual well 1000 of a microplate (such as microplate 100 depicted inFIG. 1) and immediately surrounding microplate material. In someembodiments, well 1000 that is e.g., part an open welled microplate,such as may be part of incubator system 300.

As illustrated in FIG. 10a , well 1000 has an open portion 1010. FIG.10b depicts a representation of an example separate self-contained cap1020 and associated fluidic/gas-tight seal 1030 that can be placedagainst the open portion 1010 of the microplate well 1000. FIG. 10cdepicts a variation on the arrangement depicted in FIG. 10b wherein thecap 1040 covers not only open portion 1010, but also top surface of well1000. In some embodiments, cap 1040 is not a separate self-containedstructure but instead rendered as a well or cavity of a larger piece ofstructural material.

Both of the arrangements depicted in FIG. 10b and FIG. 10c can transformthe well of a microplate (such as that depicted in FIG. 1) into aseparately sealed chamber to which provisions for one or more offluidics, gas exchange, temperature control, sensors, imaging, and otherfunctions can be dedicated. The one or more of fluidics, gas exchange,temperature control, sensors, imaging, and other functions can berendered in the microplate, as described above, or in the cap, asdescribed in pending U.S. patent application Ser. No. 13/761,142, or inboth in the cap as well as in the (also as described in pending U.S.patent application Ser. No. 13/761,142).

As an example, fluidics can be considered: In addition to (or as analternative to) microfluidics and sensors rendered within themicroplate, fluidics arrangements linking to caps mating with one ormore wells of a microplate can be provided. Additionally, the adaptedand/or modified cap arrangement (as illustrated in FIGS. 10b and 10c )can be configured with fixed sensors (for example, sensors for one ormore of temperature, O₂, CO₂, NO, CH₄, NH₃) in the cap and specialpurpose sensors (for example monitoring biomarkers, other excretedproteins, and waste products) on the removable replaceable mediaelement. The cap can also be configured to provide gas exchange,regulated thermal control, and arrangements can be used to dispenseprotective materials to prevent or fight unintended infections fromvarious phages, pathogens, parasites, and competing intruder cell types.The fluidic environment provided by the cap can also be used tointroduce materials to be exposed to the cells, for example drugs,pharmaceutical agents, photosensitizers, fluorophore probes/markers,etc.

As another example, fluidics arrangements provided by a cap can beconfigured to provide individual wells in the microplate with controlledfluidic delivery of nutrients. As another example, fluidics arrangementsprovided by a cap can be configured to provide individual wells in themicroplate a controlled fluidic delivery of dissolved gases.Additionally, fluidics arrangements provided by a cap can providecontrolled fluidic removal of waste materials so as to support lifeprocesses in one or more wells of the microplate. The fluidicsarrangements, which can also be used for gas exchange and other types ofmaterial transport (including slurries and suspensions) can include useof a controllable multichannel microfluidic chemical bus such as thattaught in pending U.S. Pat. No. 8,032,258 and pending U.S. patentapplication Ser. Nos. 13/251,286 and 13/251,288. In some embodimentsthese fluidic paths can be used to carry solvent(s), cleaning fluidsand/or clearing gases as taught in pending U.S. patent application Ser.Nos. 11/946,678 and 13/314,170.

FIG. 11a depicts a variation on the arrangement depicted in FIG. 4bwherein the centralized gas mixture operation is replaced with amultichannel gas distribution bus 1110 and controllable amounts ofselected gas components (as controlled e.g., by controller 420) aredelivered individually to each well and cap chamber. The multichannelgas distribution bus can be realized, for example, as (or using the samesystems and methods as) a controllable multichannel microfluidicchemical bus such as that taught in pending U.S. Pat. No. 8,032,258 andpending U.S. patent application Ser. Nos. 13/251,286 and 13/251,288.FIG. 11b depicts a variation on the arrangement depicted in FIG. 4bwherein separate post-distribution gas purification is provided for atleast some of the well and cap pairs.

FIG. 12 illustrates a fluid media replenishment system 1200 according tosome embodiments. System 1200 provides separately controlledenvironments, separately controlled reactant introduction, and separatemonitoring, each for an associated cell culture or group of cellcultures. Thus cell culture (or group of cultures) 1220 receives its ownseparately controlled thermal and gaseous control and reaction orsubstrate monitoring, while culture (or group of cultures) 1230 receiveits own separately controlled thermal and gaseous control and reactionor substrate. The arrangement depicted in depicted in FIG. 12 also showsthe distribution of fluidic cell culture media 1210 into cell culturewells and the passage of excess fluidic cell culture media containingcell waste products 1250 and other discards.

Embodiments can further include other of features, adaptations, andmodifications of features, arrangements, methods, and technologiesdescribed in pending U.S. patent application Ser. No. 13/761,142suitable for adding value to a cell incubator.

Additionally, in some embodiments dedicated imaging camera arrangementscan be provided for at least some of the well and cap chambers.

The incubated cells supported in the described environment can beadvantageously used in various experiments heretofore not otherwisepractical or feasible. Some example experiments include:

-   -   Controlled regulation of oxygen to invoke various types of        oxygen stress,    -   Controlled regulation of nitric oxide to study concentration        effects on cell processes,    -   Controlled dispending of pharmaceutical agents to the cells,    -   Controlled optical stimulation of native cells or cells prepared        with photosensitizers, etc.,    -   Controlled regulation of temperature and gaseous portion for        each isolated specific environmental cell growth,    -   Controlled dispending of infectious agents to the cells,    -   Controlled dispending of chemical or biochemical compounds to        the cells,    -   Controlled dispending of toxins and non-infectious agents to the        cells.

In the experiments, various sensors (and imaging cameras if used) can beemployed to monitor the status, condition, and response of the livingcells. Other types of experiments, features, enhancements modifications,and design variations are anticipated and provided for by the presentapplication.

For example, various embodiments can focus on three mainfunctions—regulating and the cultural environment, monitoring thecondition such as concentrations of the adding substances duringincubating, and analyzing the cellular responses—cell assay. Controllingcell growth conditions—temperature, gaseous portion, humidification, andsterilization—will be able to modify each factor to meet the optimalcondition for cell growth and studied designs. For example, varioustypes of cells require distinctly different environmental conditions.Additionally, in order to study the cell responses to differentconditions such as condition of oxygen levels and carbon dioxide levels,it is desirable that a new generation cell incubator be able to adjustenvironmental and other experimental factors during incubating in aprecisely control manner for different well and cap chambers. Forinstance, embodiments of the incubator can mimic hypoxia (low oxygenlevel) using a 1:19 CO₂/N₂ mixture and can mimic normoxia (normal tissueand cell culture oxygen level) using a 5% CO₂ in each cell cultureenvironment.

The process of cell culture contains mixing and washing steps. Sinceerrors from taking the cells out of the incubator for professing themixing and washing steps inducing a discontinuing the incubatedcondition may occur. The embodiment of the incubator focuses oncombining every step of the cell culture processes in one place byembodying microfluidic systems and biosensor detection to the incubator.The microfluidic systems allow the solutions to automatically mix andmove to where the cells locate during incubating.

The sensor implementation, fluidic/gas interfacing, miniaturizingapproaches, electrical interfaces and optical interfaces, and further bycollocating, and integrating a large number highly-selective sensors andchemical sensors—together with appropriately selected supplementalsensors (for example temperature, pH, selective ions, etc.)—the presentapplication provides a rich ability to flexibly perform, create, deploy,maintain, and update a wide range of panels, assay, array, and/orsequence of tests for a wide range of substances and microorganism canbe created.

Monitoring the biological process of the cells—temperature, pH, CO₂,etc.—and the levels of the studied substances, such as nitric oxide andother free radical during incubating is applied to the presentapplication. To monitor the biological processes and measure thequantity of the candidate substances, biosensor is developed and appliedto the incubator. The signal from the sample translocate through asemiconductor cell plate, which is observed. The biosensor is alsobeneficial for cell assay such as apoptotic assay and cell viabilityassays (such as the MTT assay). In addition, chemical and biologicalsolution is transferred from the solution's storage to the wells byusing microfluidic system.

The incubator comprises the multiple independently operated andcontrolled cell culture chambers. These can, for example, be arranged inthe form of an array, micro-well array, assay, etc. Cell cultureenvironmental control and sensors are applied to an individual chamber.To control the cell culture condition such as temperature and gaseousportions, each individual chamber consists of a cap, which provides aselective the cell culture condition. For example study the effects ofNO on gene expression in hypoxia condition. In this study, to operatingthis experiment, NO donors are added to the cell samples in low O₂levels culture condition and they are also added into other samples innormoxia condition as controls.

In some embodiments, each well and cap chamber comprises dedicatedsensors that are able to detect biological or chemical processes duringcell culturing.

Such features more readily facilitate the simultaneous parallel studycell cultures subjected to controlled differences in concentrations,environmental controls, and introduced substances.

Example Cell Culture Requirements

To keep cell cultures alive outside of tissues, a number ofenvironmental, nutrient, and sterility factors must be maintained.Additionally, in duce cell growth, hormones or growth factors must beprovided. A few of these aspects are considered here in the context ofvarious embodiments.

Temperature Control:

Temperature plays an important role in cell culture. For example, thelength of bacteria, L. pneumophila serotype 1 strains AATCC33152,responded to variations in temperature over a range from 30.0° C. to47.0° C. in 0.5 steps using a developed temperature gradient incubator[1]. Additionally, cells are able to survive at temperatures 10° C. to20° C. below the core temperature, but they will immediately destruct ifthe temperature reaches 40° C. [2]. In general, in the cell biologyfield, the culture temperature is 37° C. (human body temperature).However, various cell types require other cell culture conditions.Typically controlled temperature ranges of 30.0° C. to 47.0° C. would bemaintained by embodiments employing heating and cooling functions asrequired. In some applications the ability to vary temperature over arange as a function of time or as an experimental parameter isadvantageous.

Gaseous Environment:

The cell grown requires a combination of 90-95% air and approximately 5%CO₂. The partial pressure of oxygen is at 18%. However, to study theeffect on concentration of O₂ such as in hypoxia condition (lower O₂levels), incubators should contain the mixture of carbon dioxide (CO₂),nitrogen (N₂), and air to obtain the desired gaseous environment [3].Indeed, the cells also produce CO₂, which will affect to a normal pH (pH7.0-7.4) of the cell medium. Furthermore, because of requiring asterilization of the cell culture and there is a contamination from theair that might damage the cells, a filtration system such as ahigh—efficiency particulate air (HEPA) is regarded to establish in theincubator.

Humidification:

Humidification is important to control the osmolarity of the cells.Osmolality involves with the movement of the solvent through a semipermeable membrane into a solution which has higher concentration ofsolute. If the compressed air in the incubator has really low moisturecontent, it will cause evaporation of the cell medium and increaseosmolality. Consequently, the cells will lose water and shrivel. On theother hand, at high moisture content, a cell will gain too much waterand can become lysed. Accordingly, it is useful for some embodiments toincorporate a controlled humidification will be applied to the internalchamber of the incubator without losing water due to the movement of thedevice. Alternatively, a water tray or similar arrangement thatfacilitates intake of water through ports and, for example, saturates apad at the bottom of the system, regulates the humidity. The flow of thegaseous mixture draws moisture from the pad which will maintain thehumidification of the environment. Embodiments can employ knowtechnologies such as evaporation and condensers to regulate humidity ofgases. In some embodiments, thermoelectric devices can be used to createelectrically-controlled cooled surfaces for condensing moisture from airand gas mixtures and heated surfaces for condensing moisture from airand gas mixtures.

Sterility:

Sterility plays in an important role in cell culture. Thus, theincubation apparatus needs antimicrobial environmental condition, forexample employing appropriate amplitudes and wavelengths of UV light inappropriate settings so as to kill ambient bacteria. In the case ofunintended bacterial infection of an active cell culture, agents such asantibiotics or bacteria phages can be useful to save the life of thecells in the infected culture.

Gentle Fluid Administration

As discussed earlier in conjunction with FIG. 12, various embodimentscan advantageously provide distribution of fluidic cell culture mediainto cell culture wells and the passage of excess fluidic cell culturemedia containing cell waste products and other discards. Additionally,various embodiments can advantageously provide reagents such ashormones, signaling donors, stains, etc. as well as other materials suchas drugs, dissolved gases, infectious agents, antibiotics, etc. whichcan be introduced into cell culture wells by fluids.

FIG. 13 depicts an example arrangement 1300 wherein cell culture mediafluid 1310 flows from a fluid source to individual cell culture wells1320 and from there to fluidic waste handling 1330, and where reagents1340 such as hormones, signaling donors, stains, etc. can be introducedinto cell culture wells 1340 by fluids. A similar arrangement can beused for fluidically introducing other materials such as drugs,dissolved gases, infectious agents, antibiotics, etc. into cell culturewells. In some embodiments, the flows of media fluid 1310 areindividually controlled for each microplate, e.g., by controller 420. Insome embodiments, the flows of media fluid 1310 are individuallycontrolled for groups of wells within a specific microplate, e.g., bycontroller 420. In some embodiments, the flows of media fluid 1310 areindividually controlled for each well, e.g., by controller 420.

In some embodiments, care is taken in the introduction of the fluidics1310 into the cell culture wells 1320 so that the forces exerted byfluid currents do not damage cell adherences or other physical aspectsof the cell culture. One approach involves using extremely low rates offluid flow. Another approach can also help uniformize the distributionof new fluidic material over the surface of a well 1320. This approach,illustrated in FIGS. 14a and 14b , involves gentle fluid flow over aramped circular flow arrangement around a rim of a well.

One approach to individualizing the rate and contents of flow deliveryis by connecting such arrangements to an instance or adaptation ofcontrollable microfluidic bus technologies 1510, such as those taught inU.S. Pat. No. 8,032,258 and pending U.S. patent application Ser. Nos.13/251,286 and 13/251,288), for example as depicted in FIG. 15.

In some embodiments, fluids can be introduced from below the cell wells,as depicted in FIG. 16a . In other embodiments, fluids can betransported in and out of individual cell culture wells 1630 bysurface-exposed trays 1620, for example as depicted in FIG. 16b , orfrom the edges of the well sides. Combinations of these approaches canalso be used, for example as depicted in FIG. 16 c.

Temperature Control

Various methods can be used to control the temperature of group of cellculture wells and individual cell culture wells, for example:

1. Control of gas environment temperature2. Control of media fluid temperature3. Control of microplate material temperature4. Control of individual well wall temperature.

Approaches 1-4 can be accomplished, for example, with controlledthermoelectric devices (providing cooling and/or heating) as well aselectrical resistive heating elements. Approach 1 can also employrefrigeration techniques.

Approaches 3 and 4 are also particularly suitable to fluidic heating andcooling methods employing associated fluidics throughout a microplate.Thus systems and methods for the temperature control of fluids canaddress Approaches 2-4. As to systems and methods for the temperaturecontrol of fluids, self-contained approaches can use controlledthermoelectric devices to providing cooling and heating, orthermoelectric devices to providing cooling and electrical resistiveheating elements for heating. In some embodiments, two reservoirs offluids, one cooled and the other heated, are used and mixed in variousproportions to obtain the desired fluid temperature.

Higher precision in temperature control can be obtained by employingthermal insulation among wells, on microplate surfaces, etc. and well asthrough the use of temperature sensors driving feedback controllers, forexample employing simple on/off control or continuous-range controlusing for example PID (“Proportional/Integral/Derivative) feedbackcontroller systems and/or algorithms.

Sensors and Detection for Cell Culture Wells and Well/Cap Chambers

The microprocessor-controlled microfluidic-based incubating platform forcells can be configured to comprise removable replaceable media elementcomprising an array of sensors. These sensors can include for exampleelectrochemical, bio-FET, and optical sensor technologies. In variousembodiments sensors can configured to measure concentrations of gases,chemical substances, proteins, biomarkers, ions, etc. as well as otherquantities such as temperature and optical properties.

This section briefly describes a number of existing and emergingbiosensor and chemical sensor technologies and approaches suitable oradaptable for full microsystem implementation. The synergistic use ofexisting biosensor and chemical sensor technologies and approaches, anumber of adaptations others and addition sensor innovations, plus yetother adaptations and innovations, as employed in the presentapplication will provided in later discussion.

One simplified representation of a unified view of the basis ofbiosensing is provided in FIG. 17a . A sample or analyte 1710 is broughtinto interaction, communication, and/or physical contact, with arecognition process 1720. In general, the recognition process 1720internally employs a selective detection material or process suchmembranes, enzymes, antibodies, cells, molecular imprint materials,electron orbital transitions, magnetic resonances, etc. The recognitionprocess 1720 results in an observable or measurable effect that is inputto a transduction process 1730 (comprising, for example, one or more ofan electric field, optical, chemical, magnetic, electric current,electric voltage, etc.) to an output signal process 1740 (which maycomprise one or more field effect transistors, electrochemical electrodearrangements, photo-responsive electric devices, magnetic-responsiveelectric devices, etc.), typically producing an electrical signal 1750.The many components of each class (distinguished as columns in theFigure) can be arranged in various combinations to form an extensiveplethora of sensing approaches, systems, methods, and devices. Somesensing approaches can include more than one choice from each class—forexample, an enzyme cascade could be used, and in one example embodimentof the present application to be discussed, living cells may be used toprovide front-line recognition processes, and materials secreted throughthe membranes of the living cells can be subjected to at least asecond-line recognition process (employing for example one or moreenzymes, antibodies, molecular imprinted materials, etc.).

In particular there are a rapidly increasing number and diversity oftechnologies and approaches for chemical sensor and biosensor that aresuitable or adaptable for microsystem implementation. Although notcomprehensive or exhaustively or precisely organized, FIG. 17b providesa relatively comprehensive view of biosensors and approaches suitable oradaptable for microsystem implementation. Many of these sensortechnologies and approaches are still either being prototyped inrelatively large sizes, mostly for the convenience of inexpensive andflexible construction in a traditional laboratory. Many others arecurrent implemented as small structures supplemented with largelaboratory instruments and devices that can be simplified, focused,specialized, adapted, or otherwise miniaturized. Broadly these can beclassified into at least the following electronic device and operationcategories:

-   -   Electrochemical sensors 1750;    -   Organo-Electrochemical Transistor (OECT) sensors 1760;    -   Bio-FET sensors 1770;    -   Optical sensors 1780 (these to be adapted to comprise        opto-electrical devices), and these can include at least the        following active sensing agents and sensing components:    -   Molecular imprint materials (“MIMs”);    -   Antibodies (as well as synthetic antibodies);    -   Enzymes (as well as other proteins, synzymes, etc.);    -   Photo-responsive, photo-absorption, and photo emission        materials.        Various configurations and arrangements of these in turn can        function as “biosensors,” “immunosensors,” “chemical sensors,”        etc. These and other relevant sensing technologies are taught in        pending U.S. patent application Ser. No. 13/761,142.

A brief overview of non-imaging sensing is provided below. (As indicatedabove, imaging sensors can also be included for use with a removablereplaceable media element; these will be discussed later.)

Electrochemical, BioFET, and ChemFET Sensing Methods

Classical, contemporary, and advancements in electrochemical sensors areknown. A few remarks regarding aspects of current and emergingelectrochemical sensors relevant to various aspect of the presentapplication are made in this section.

There are various major types of electrical sensing process responsiveto chemical conditions and processes that are employed inelectrochemical sensors, for example:

-   -   “Potentiometric electrochemical sensors” involve measuring the        difference between two potentials (in units of volts) associated        with the electrodes of an electrochemical sensor,    -   “Amperometric electrochemical sensors” involve measuring current        (in units of amperes) through an electrochemical sensor,    -   “Conductometric electrochemical sensors” (also referred to as        “chemiresistors”) involve measuring the “direct-current” (DC)        resistance (in units of ohms) or conductance (in units of mhos)        across an electrochemical sensor (resistance being the ratio of        voltage to current and conductance being the ratio of current to        voltage),    -   “Impedance electrochemical sensors” involve measuring the        sinusoidal alternating current (AC) reactance, either as        impedance (in units of ohms) or admittance (in units of mhos)        across an electrochemical sensor over an adequate range of AC        frequencies.

Also of importance is a means, process, material, or other arrangementproviding adequate (or useable) selectivity of the sensors response tochemical or biochemical substances of interest with respect to expectedrange of chemical constituents in a sample. In some cases, sensors canbe made very selective (for example, an antibody-based electrochemicalsensor employing an antibody that responds only to a specific protein)or selective to a family of materials and thus in some applicationsrequiring strict limitations on what can be in an applied sample.Examples of such means, processes, materials, and other arrangementsinclude uses of membranes, specialized crystals, enzymes, and antibodiesamong many other approaches, and can include combinations of multiplemeans, processes, materials, and other arrangements. For an extensiveexamples of what types of quality chemical and biochemical detectionsthat can be accomplished with simple means, processes, materials, andother arrangements for the family of simple 3-electrode electrochemicalsensors comprising simple carbon paste electrodes, the reader mayconsult the extensive tables in the book by I. Svancara, K. Kalcher, A.Walcarius, K. Vytras, Electroanalysis with Carbon Paste Electrodes, CRCPress, 2012, ISBN 987-1-4398-3019-2 and the techniques and applicationsdiscussed in the book by Raluca-Ioana Stefan, Jacobus Frederick vanStaden, Hassan Y. Aboul-Enein, Electrochemical Sensors in Bioanalysis,Marcel Dekker, 2001, ISBN 0-8247-0662-5.

The means, process, material, or other arrangement providing adequate(or useable) selectivity further typically employs an associatedlimitation on the sample applied to the sensor. For example, somesensors approaches are relevant only to dry gases, others relevant onlyto liquid samples, while others relevant to more complex samples such assuspensions (for example comprising cells), gases dissolved liquids,materials at thermodynamic critical points (such as vapors and gasesincluding vapors), slurries, gases comprising particulates or colloids,emulsions in various stages (flocculation, creaming, coalescence,Ostwald ripening, etc.), micelles, etc. as well as combinations ofthese.

Regarding miniaturization, it is noted that electrodes whose diameter issmaller than 20 μm (“microelectrodes”) provide best performance asamperometric chemical sensors. Additionally, in the miniaturizationpotentiometric ion sensors, a chemical species-selective membrane isplaced directly on (or used as) the insulator of a Field EffectTransistor (FET) input gate terminal, resulting in a miniaturizedchemically selective field-effect transistor (CHEMFET) or ion-sensitivefield-effect transistors (ISFET) It is noted that the miniaturization ofthe reference electrode compartment within a potentiometric ion sensorlimits its operational lifetime. However, aspects of the presentapplication prevent the need for long operational lifetimes and thislong standing limitation and concern can be de-emphasized.

Electrochemical impedance spectroscopy (EIS), also referred to orassociated with Dielectric Spectroscopy (DS) and Impedance Spectroscopy(IS), measures the electrical impedance of an analyte over a range offrequencies. The electrical impedance is responsive to the dielectricpermittivity properties of the analyte which due to the electric dipolemoment interaction with time-varying imposed (usually electrical)fields. In contrast to the voltammetry and amperometry electrochemicalsensors described above (which involve measurement of DC or pulsed-DCelectrode current as a function of applied electrode-solution voltageand rely on changing in electrode conditions), impedance sensors measurethe electrical impedance by imposing a small AC voltage between sensorelectrodes over a series or swept range of frequency and measuring theresulting AC current. As frequency increases the dominatingelectrochemical processes evolve through regimes of ionic relaxation,dipolar relaxation, atomic resonances, and electronic resonances athigher energies.

An emergent subclass of electrochemical transducers areOrgano-Electrochemical Transistor (OECT) sensors employingimmuno-recognition materials. Examples of these have been constructedthat claim 1 ppm sensitivity. OECT sensors can operate in at least twodifferent mechanisms:

-   -   Doping/Dedoping effects, for example where an antibody        immobilized on the surface of a Field Effect Transistor gate        channel surface binding to a charged ligand, the resulting fixed        local charge that attenuates ion diffusion into the channel,        thus altering the channel conductivity.    -   Antibody conformational changes, for example where an antibody        is incorporated into a channel whose conductivity is affected by        conformational changes in antibody that are induced by ligand        binding.

Classical, contemporary, and advancements in “bioFET” sensors are known.An example is an ion-selective field effect transistor (“ISFET”). MostISFETs employ an analyte solution as the gate electrode of theField-Effect Transistor (FET), while the source and drain of the ISFETare as those of a typical Metal-Oxide Semiconductor Field-EffectTransistor (MOSFET). The gate insulator, typically made employing SiO₂,Si₃N₄, Al₂O₃ and Ta₂O₅), can be affixed or otherwise modified to includeor attach ion-selective substances. The selective activation byassociated ions affects the electric fields presented to the gateinsulator, in turn varying the current through the FET channel. Such asensor can be used to sense pH and concentrations of various chemicalcompounds that affect the operation of sensors in a larger systemexamining the same sample. Further, additional materials and layerstructures can be attached which comprise bio-selective materials that,when selectively activated by associated biomolecules, create ions thatare measured by the ion-selective sensor. In order to miniaturize someISFET arrangements, the depicted reference electrode becomes impracticaland/or a limitation—for example due to issues of relative physical sizeand active-use aging—and Reference Field Effect Transistors (REFET) areemployed instead. However these, too suffer from various limitations,including thermodynamic equilibrium, recalibration needs over the sensorlifetime, and other active-use aging issues. As will be seen, theclassical concerns for reference electrodes and REFETs are evaded by theusage and operational modalities employed in the present application tobe disclosed. Other versions incorporate highly-selective materials orother layer structures that comprise bio-selective substances that areselectively activated by associated biomolecules in a manner thataffects the conductivity or induced electric fields presented to thegate insulator, in turn varying the current through the FET channel.

An aspect relevant to various embodiments is the fact that many of theelectrochemical and Bio-FET sensors can be created from layered stacksof materials. Further, the materials employed in such sensors can befunctionally replaced with entirely other types materials (for example,organic semiconducting and conducting polymers) that can beinexpensively “printed” via so-called “Printed Electronics” and“Functional Printing” manufacturing technologies using fancierindustrial-scale forms of ink-jet printers. The present applicationexploits such “Printed Electronics” and “Functional Printing”manufacturing technologies (as will be discussed later).

Optical (Non-Imaging) Sensing Methods

Classical, contemporary, and advancements in optical markers, opticallabels, and optical sensors relevant to biological analysis are known. Afew remarks regarding aspects of current and emerging optically-baseddetection technologies relevant to various aspect of the presentapplication are made in this section.

In most contemporary laboratory instruments, space-consuming expensiveprecision optical elements, such as diffraction gratings with precisealignments to photodiode arrays, are employed. However, an aspectrelevant to the present application is, (as taught in Pending U.S.patent application Ser. No. 13/761,142) that many types ofoptically-based detection technologies such as those employed inmicroplate/microarray technologies and techniques can be modified oradapted for useful miniaturized implementation comprising at least someportions having layered structures suitable for fabrication by printing.Most optical sensing techniques employing optically-based technology forbiochemical applications have been developed in the product andtechnology context of large laboratory instruments, and thus thecomprehensive miniaturized implementations used in various embodiments,for example such as those in Pending U.S. patent application Ser. No.13/761,142, differ from current trends in industry and academicresearch. For example, some of the modifications and adaptations to bepresented leverage small ultraviolet LEDs, while other modifications andadaptations leverage a family of wavelength-selective LED based sensingtechnologies, such as those taught in Pending U.S. patent applicationSer. No. 13/761,142 which remove with the need for large and/orexpensive precision optical components and precise alignment needsrequiring expensive manufacturing processes.

As to optical detection involving the emission of light, an importantexample of optically-based technology for biochemical applications isthe use of fluorophores (also called fluorochromes) which absorbexcitation light of a first wavelength (usually ultraviolet or visiblelight), attain an electronic excited state, and as the excited statedecays emit light at a second (lower-energy, longer) wavelength,typically arranged to be in the visible (or in some cases, infrared)light range. Fluorophores are used as staining dyes for tissues, cells,enzyme substrates, etc. and used as a probe or indicator (when itsfluorescence is selectively affected by effects of species polarities,proximate ions, excitation light polarization, etc.) and can be arrangedto covalently bond to a biological molecule (such as enzymes,antibodies, nucleic acids, and peptides) so as to optical mark thelocation and presence or activity of that biological molecule.Fluorophores can be used to mark cells, structures or materials withincells, and in conjunction with antibodies and other selective ormodulating agents in microarrays. Although most fluorophores are organicsmall molecules, it is noted that fluorophores size can stericallyaffect the biological molecule it is used to tag, as well as othereffects. It is also noted that solvent polarity can affect fluorescenceintensity.

Another optical detection involving the emission of light arechemiluminescence tags and labels. The origin of emitted light fromchemiluminescence processes is distinguished from the fluorescenceprocesses of fluorophores in that the electronic excited state producingemitted photons result from a chemical reaction instead of excitation byincoming light. One example is luminal (C₈H₇N₃O₂) which is employed inmicroarray, assays, and other detection of copper, iron, cyanides, andspecific proteins by Western Blot.

Further, in measuring at least fluorophore light emission, there are atleast two measurement techniques that can be made and used in markingstrategy design. The first of these is measuring of the formal lightamplitude or formal light intensity of the fluorophore emissions,usually spatially normalized (for example per observational unit volumeof sample, per unit area of an observational field, etc.), andnormalized with respect to background levels or other factors. Thesecond of these is the measurement of fluorescent lifetime whichtypically are effectively unaffected by probe concentration, excitationinstability, photobleaching, washout, and other phenomena complicatingamplitude and intensity measurements. Since fluorescent decay times arein the range of 1-20 ns, short excitation pulses, high-speed opticalsensors, and radio-frequency electronics can be required. Alternatively,phase modulation techniques, such as those described by H. Szmacinskiand J. Lakowicz in the article “Fluorescence Lifetime-based Sensing andImaging,” Sensors and Actuators B: Chemical (Proceedings of the 2ndEuropean Conference on Optical Chemical Sensors and Sensors), Volume 29,Issues 1-3, October 1995, pp. 16-24 and earlier book chapter“Lifetime-based Sensing Using Phase-Modulated Fluorometry” inFluorescent Chemosensors for Ion and Molecule Recognition, AmericanChemical Society, 1993, ISBN 0-8412-2728-4, Chapter 13, pp. 197-226.Additional fluorescence sensing technologies and methods of value inincorporating into the present application include time-resolvedfluorescence detection and measurement techniques responsive tofluorescent polarization and anisotropy phenomena,

Each of the two above optical detection arrangements involve emission oflight, but optical-based detection can also leverage absorption oflight, for example employing colorimetry and photospectroscopy. Oneimportant example of this is Enzyme-Linked Immunosorbent Assay (ELISA)technologies that employ enzymes (as well as antibodies or otherselectively responsive agents) to invoke visual color changes responsiveto the presence of a target material. An example specialized productarea employing these is the ArrayTube™ technology comprising avertically-oriented reaction vessel arranged with a (non-fluorescent)colorimetric array at the vessel bottom. An example ‘selection-guide’treatment comparing fluorescent, chemiluminescent, and colorimetricdetection schemes and agents can be found in Selecting the DetectionSystem—J. Gibbs, Life Sciences “Colorimetric, Fluorescent, LuminescentMethods,” ELISA Technical Bulletin—No. 5, Corning Incorporated, 2001 (asdisclosed at world wide web atcatalog2.corning.com/Lifesciences/media/pdf/elisa5.pdf, visited Jan. 27, 2013).Analogous to the fluorophore, the moiety responsible for the color of amolecule is called a chromophore.

A great many fluorophores and chromophores are permanently active(albeit modulated by solvent polarity, pH, temperature, etc.) and do notchange their emission or absorption properties as a result of anybinding event. Such markers simply tag molecules such as enzymes andantibodies and variations in emission or absorption properties of thesample or parts of the sample result from changes in spatialconcentration of enzymes, antibodies, etc. as they cluster in theirbinding within localized regions of antigen. Other fluorophores andchromophores are or can be configured to change their lightemission/absorption properties in direct response to binding events—forexample as with calcium markers. Addition performance considerations canbe considered, for example whether the fluorophores and chromophores areintrinsic or extrinsic as considered in T. Bell et al., “IntrinsicChromophores and Fluorophores in Synthetic Molecular Receptors,” inFluorescent Chemosensors for Ion and Molecule Recognition, AmericanChemical Society, 1993, ISBN 0-8412-2728-4, Chapter 7, pp. 85-103.Related techniques of value to the present application includefluorescent probes that indirectly sense analytes via chemicalreactions, for example but not limited to “turn-on” fluorescent probesdiscussed for example in M. Jun, B. Roy, K. Ahn, “Turn-on fluorescentsensing with reactive probes,” Chem. Commun., 2011, Issue 47, pp.7583-7601.

DNA-oriented microarrays (also called “DNA chips” and “biochips”)comprise small DNA regions arranged in an array on a plate material, andare used to simultaneously measure gene expression levels of manysamples or tests in parallel, genotyping of genome regions, etc.employing fluorophores, chemiluminescent, or other types of labels ortags.

Protein-oriented microarrays employing fluorophores are widely used foridentification, characterization, and study of disease biomarkers,protein-protein interactions, specificity of DNA-binding and proteinvariants, immune response, etc. These methodologies provide an importantcontemporary tool for next-generation understanding of cell biology,disease, and drug development as explained, for example, in C. Wu,(ed.), Protein Microarray for Disease Analysis: Methods and Protocols,2011, ISBN 1617790427, or in the handbook provided by AmershamBiosciences entitled Fluorescence Imaging: Principles and Methods, 2002(document 63-0035-28 Rev.AB, 2002-10, as disclosed at world wide web atcancer. duke.edu/DNA/docs/Phosphorimaging%20_%20Fluorescent_Scanning/Fluorescence%20Imaging%20Handbook.pdf,visited Jan. 26, 2013). In addition to their use in biochemical samples,they can also be used in living cells to monitor cell metabolism andcell signaling, for example as with “Fluo-Calcium” indicators and in thetechniques described in R. Wombacher, V. Cornish, “Chemical tags:applications in live cell fluorescence imaging” J. Biophotonics 4, No.6, pp. 391-402 (2011).

Accordingly, the present application can leverage adaptations of thistechnology base so as to provide support for applications involvingmeasure gene expression levels of many samples or tests in parallel,genotyping, next generation understanding of cell biology, disease, anddrug development. Of relevance to the adaptations made in the presentapplication to be described is that the ranges of light wavelengths forexcitation emission are those of commercially manufactured LightEmitting Diodes (LEDs), and, as explained, that LEDs of differingemission wavelengths can be used as wavelength-selective detectors astaught in Pending U.S. patent application Ser. No. 13/761,142.

Recognition by Antibody, Synthetic Antibody, and Related Materials

As alternatives to animal-produced antibodies, various embodiments alsoprovide for recognition by antibody, synthetic antibody, and relatedmaterials as taught in Pending U.S. patent application Ser. No.13/761,142

Recognition by Molecular Imprinting Materials (MIMs)

As described above, biochemical materials and approaches that can beemployed as alternatives to animal-produced antibodies for sensors usedin the present application include synthetic and recombinant antibodies,recombinant antibody fragments, synbodies and unstructured peptides. Incontrast to all of these, Molecularly Imprinted Material (MIM)technologies, such as Molecularly Imprinted Polymers (MIPs), leveragesynthetic materials as an alternative to antibodies in highly selectivesensors. MIMs can be used to recognize and bind to a target moleculewith high affinities and specificities that can rival antibodies,receptors, and enzymes.

Molecularly Imprinted Polymers (MIPs) can be inexpensively andreproducibly manufactured by polymerizing commercially availablemonomers in the presence of a templating molecule structurally similarto a specified target molecule. Because MIPs are heavily cross-linked,and thus cannot experience conformational rearrangement, MIPs providefar superior stability to biological antibodies, offering considerablylonger shelf-life, less stringent storage requirements, and can be usedwith extreme pH, temperature, ionic strength, and other operatingconditions outside that of most antibodies. A representative review isprovided in L. Ye, K. Mosbach, “Molecular Imprinting: SyntheticMaterials As Substitutes for Biological Antibodies and Receptors,”Chemistry of Materials, 2008, 20, pp. 859-868). MIMs still fall short inmatching or exceeding the specificity and cross reactivity rejection ofbiological antibodies, and this has been viewed as a problem indiagnostics because of higher probabilities of false positives. However,various aspects of the present application's methodology, architecture,and statistical processing approaches provided for by the presentapplication can inherently significant diminish this concern.

As sensors relevant to the present application, one of manyrepresentative reviews and summaries regarding the use of MIMs and MIPsas selectivity agents in sensors is provided in G. Guan, B. Liu, Z.Wang, Z. Zhang “Imprinting of Molecular Recognition Sites onNanostructures and Its Applications in Chemosensors,” Sensors, 2008, 8,pp. 8291-8320. Of additional utility to the present application is thefact that MIMs have demonstrated robust liquid and gas chemical sensorsfor more than a decade (see for example F. Dickert, O. Hayden,“Molecular Imprinting in Chemical Sensing,” Trends in AnalyticalChemistry, vol. 18, no. 3, 1999).

Recognition by Other Recognition Materials

Many other types of selective detection materials can be used by thepresent application, including peptides, genetically engineeredproteins, carbohydrates, nucleic acids, oligonucleotides, amtamers,phages, and even living cells and tissues cultured from plants andanimals. A representative survey of such additional types of selectivedetection materials that can be employed by the present application canbe found in the extensive book M. Zourob, (ed.), Recognition Receptorsin Sensors, Springer, 2010, ISBN 978-1-4419-0918-3.

Various embodiments can employ several component core, design, andfabrication technologies including:

-   -   Organic semiconducting and conducting polymers,    -   Printed electronics and functional printing,    -   Microfluidic systems and their fabrication,    -   A range of currently experimental sensor technologies that have        been or can be adapted for microfluidic use,    -   Rapidly-advancing commercial production of a wide ranges of        highly selective antibodies and enzymes,    -   Laboratory methods and analysis, together with associated        biochemistry, for pathogen detection,    -   Reconfigurable microprocessor-controlled Lab-on-a-Chip (RLoC)        technologies (U.S. patent application Ser. Nos. 11/946,678 and        13/314,170),    -   Microfluidic chemical bus technologies (U.S. Pat. No. 8,032,258        and pending U.S. patent application Ser. Nos. 13/251,286 and        13/251,288),    -   Microfluidic and Lab-on-a-Chip development technologies (U.S.        patent application Ser. Nos. 12/328,726 and 12/328,713).

FIG. 18 illustrates a system 1800 employing underlying componenttechnologies 1810 such as:

-   -   Molecular imprinting,    -   Selective/sensitive antibodies,    -   Fluorescent indicators,    -   Optical sensor techniques and arrangements,    -   Electrochemical sensors (amperometric, potentiometric,        conductometric, membrane, diffusion barrier, etc.),    -   BioFETs,    -   Microfluidics (valves, conduits, microreactors),    -   Printed electronics,    -   Printed chemical deposition,    -   Other types of sensors,        and mid-level component technologies 1820 such as:    -   Selective/sensitive antibody-based sensors and chemical sensors,    -   Enzyme-based sensors and chemical sensors,    -   Molecular imprint sensors and chemical sensors,    -   Optofluidic devices,        and higher-level component technologies 1830 such as:    -   Selective/sensitive pathogen sensors,    -   Selective/sensitive biomarker sensors,    -   Selective/sensitive toxin sensors,    -   Selective/sensitive chemical sensors.

Overall Architecture

By unifying the sensor implementation, fluidic/gas interfacing,miniaturizing approaches, electrical interfaces and optical interfaces,and further by co-locating, and integrating a large numberhighly-selective sensors and chemical sensors—together withappropriately selected supplemental sensors (for example temperature,pH, selective ions, etc.)—various embodiments can be configured toprovide a rich ability to flexibly perform, create, deploy, maintain,and update a wide range of panels, assay, array, and/or sequence oftests for a wide range of substances and pathogens.

As to implementing the platform in a universal context to a wide rangeof applications, FIG. 19 depicts an example representation of thesynergistic and adaptive framework 1900 to create a flexiblemultiple-purpose cell incubation platform technology with vastlyexpanded capabilities. Framework 1900 includes a range of detectiontarget types 1910, such as pathogens, biomarkers, bio-toxins, andchemicals. Framework 1900 includes a range of detection agents 1920,such as absorption spectra, fluorescent marker, molecular imprint andanti-bodies. Framework 1900 includes a range of miniature detectionelements 1930, such as electrochemical detection elements, opticalelements, FETs, and fluorescent markers. Framework 1900 includes acomputing environment 1940 (e.g., a software environment) forcontrolling, analyzing, reporting, networking and for providing a userinterface.

FIG. 20 depicts an overall overview of computing environment 1940according to some embodiments. Computing environment 1940 may includesoftware, signal input hardware, signal processing hardware, andsoftware-control hardware, and may contain a memory 2010 to store one ormore of: operational algorithms, configuration algorithms, proceduralalgorithms, pattern analysis algorithms, test algorithms, analysisalgorithms and reporting algorithms. Computing environment 1940 mayfurther include one or processors to execute the one or more algorithmsstored in memory 2010. Computing environment 1940 further includesinterface electronics to interface with one or more sensors, such asBioFET Sensors, electrochemical sensors, OECT sensors, chemical sensors,optical sensors, etc.

In FIG. 20 the software is depicted at the top (signifying the softwareis oriented as being closer to the user), while the signal, sensor, andfluidic hardware is in the lower portion of the figure (signifying theseare oriented as being closer to the analyte being analyzed).

Technologies and materials applicable to embodiments of the presentapplication will continue to evolve over time. Accordingly employing theapproaches taught throughout, various embodiments can be structured toanticipate a wide range of evolutions and development in technology,techniques, protocols, usage, and applications.

Updatable software is one easily-met aspect of this goal that can bereadily incorporated, but updating of sensors, reagents, and otheraspects is far more challenging.

Removable Replaceable Media Element Approach to Microplates ComprisingSensors

According to some embodiments, printed-sensing removable replaceablemedia element approach taught in Pending U.S. patent application Ser.No. 13/761,142 can be applied to microplates comprising sensors.

Various embodiments provide a removable, practical, inexpensivelymanufactured replaceable medium providing wells for isolated cellincubation that can include a wide spectrum of low-cost sensors andreagents and memory for software. The removable medium approach providesopportunities to address a number of other issues including life-cycleand disposal, and the broader system design readily facilitatesextensions into a wide range of broader applications immediatelyspanning into health care and industrial applications.

On the logistics side, there will always be new types of testingmethodologies and improvements that are difficult if not completelyimpossible to predict. Although software changes to address aspects ofthis degree of variability and uncertainty can be provided by variousmethods, the variability of the types of physical sensors and associatedtesting reagents necessary requires some way of physically updating atleast some aspects of a testing device. Further, at least some of thesensors employed will have limited lifetimes (for example, antibodiesand enzymes could degrade) and be subject to contamination after one ormore uses.

Additionally, the removable replaceable media element can include atleast reagents.

Additionally, the removable replaceable media element can include serialnumbers, sensor specifications, and perhaps software.

The removable replaceable media further can provide an open architecturefor both third party innovation and an evolution ability. Such an openarchitecture allows for third-party development that can address a widerrange and greater number of markets (both large and small). Further, theopen architecture also allows for easy incorporation of sensortechnology improvements, and also increases the opportunity for improvedand simplified operation by users of such devices.

The technical features, value proposition, and market considerationsboth give rise to and require inexpensive mass manufacturing anddistribution. These in turn give rise to the need for the removablereplaceable medium to comprise inexpensive materials that arestraightforwardly and inexpensively assembled. An initial solution tothis is to:

-   -   Use an inexpensive substrate for the removable replaceable        medium such as some type of polymer or plastic. In various        implementations, the substrate can be rigid or can be flexible.    -   Employ functional printing (such as inkjet-printed functional        polymers deposited directly onto the inexpensive substrate) for        manufacturing the sensors and electrical aspects:        -   Printed electrodes (using organic polymer conductors)        -   Printed sensors (comprising insoluble and/or protected            layers of semiconducting polymers, materials comprising            enzymes/antibodies, deposition layers of enzymes/antibodies,            etc.)        -   Printed transistors (comprising layers of semiconducting            polymers and organic polymer conductors) for electronics.    -   Employ functional printing for manufacturing of reagent        reservoirs (for example in the form of depositions soluble        solids or gels)    -   Employ functional printing for manufacturing of Read-Only Memory        (“ROM”) (for example in the form of printed optical codes such        as printed optical bar codes, printed optical matrix codes,        printed holographic codes, printed magnetic code stripe, printed        electronic data memory, etc.)    -   If optical sensing is used, the inexpensive substrate could be,        for example:    -   Engineered to transparent for light pass-through at the needed        wavelengths, and/or    -   Employ functional printing of a optically reflective layer        (reflective at the needed wavelengths)

Further, the removable replaceable medium element and its contents canbe designed and configured to facilitate recycling, bio-hazardneutralization and/or controllable degradation facilitated by a“termination solvent” degradation-initiation fluid, etc. In someembodiments, the removable replaceable medium element comprises an arrayof sensors on a substrate. In another embodiment of the presentapplication, the removable replaceable medium element additionallycomprises electrical conductors. In another aspect of the presentapplication, the removable replaceable medium element additionallycomprises sensor interface electronics.

In some embodiments, the removable replaceable medium elementadditionally comprises data storage ROM.

In some embodiments, the removable replaceable medium elementadditionally comprises read/write data storage.

In some embodiments, the removable replaceable medium elementadditionally comprises deposits of at least one reagent.

In some embodiments, the removable replaceable medium element comprisespassive fluidics for transport of one or more liquids, gases,suspensions, slurries, etc.

In some embodiments, the removable replaceable medium element comprisesfluidics elements forming at least part of a valve for controlling theflow of one or more of liquids, gases, suspensions, slurries, etc.

In some embodiments, the removable replaceable medium element comprisesfluidics elements forming at least part of a pump inducing the flow ofone or more of liquids, gases, suspensions, slurries, etc.

FIG. 21 depicts an example abstract representation of a removablereplaceable media element 2100 comprising a plurality of cells 2110,which may be loaded with reagents, loaded with sensors or unloaded.Removable media element 2100 further includes a memory 2120, such asRom, to store data and one or more algorithms.

FIG. 22a illustrates a system 2200 comprising a removable element 2210that is configured to fit inside or attach to a base unit 2250comprising at least microfluidics 2260, a microprocessor 2280, variouselectronics 2270, opto-electronics 2290 for optical sensing, and sampleacquisition arrangements (as well as power sources, housing, EMIshielding, fluid reservoir(s), any user-operated controls, networkinterfaces, computer interfaces, visual display elements, etc.).

As another example representation, FIG. 22b depicts a simplifiedhigh-level combined signal-flow and fluidic-flow representation of oneexample of many possible implementations of the present application.This representation emphasizes abstracted hardware and transactions withthe removable element. Further details with respect to FIGS. 22a and 22bcan be found at least in Pending U.S. patent application Ser. No.13/761,142.

As other example embodiments, FIGS. 23a-23c depicts simple high-levelrepresentations of examples of systems that provide many possible userexperience and interface implementations. Each system comprises a baseunit that is implemented so as to accept and support at least, but oftenmore than one, removable replaceable media element.

For example, FIG. 23a depicts an arrangement that comprises base systemcomprising an internal user interface 2320 and other structures 2330,such as microfluidics, electronics and computational microprocessor.Internal user interface 2320 can comprise, for example software,user-operated controls, visual display elements, etc. Replaceableremovable media element 2310 is adapted to couple with base system, asillustrated in FIG. 23 a.

FIG. 23b depicts a variation on the example arrangement of FIG. 23awherein either or both of a computer interface 2360 (USB, Bluetooth, IR,etc.) and/or network interface 2350 (wireless LAN, wireless WAN,cellular, cabled-LAN, telephone land-line, etc.) is also provided. FIG.23c depicts a variation on the example arrangement of FIG. 23b whereineither or both of a computer interface 2390 (USB, Bluetooth, IR, etc.)and/or network interface 2390 (wireless LAN, wireless WAN, cellular,cabled-LAN, telephone land-line, etc.) is also provided, but in thisexample there is no internal user interface. Many variations on theseexamples are of course possible.

Additionally, the networking capabilities provide for a wide range ofpractical and expansion capabilities such as (a) download of softwareupgrades additional algorithms, and databases, (b) remote operation, (c)accessing more powerful computing for more complex data analysis, (d)interconnecting embodiments of the present application with labequipment, (e) interconnecting at least the computing environments oftwo or more instances of embodiments of the present application so thatthey can collectively act as a single larger unit in various ways, (f)remote testing, as well as other functions.

FIGS. 24a-24d depict example representations of various exampleembodiments of the removable replaceable media elements. The examplesshown here comprise example arrays of indented “wells” that can be usedas isolated cell culture sites along with other example features.

FIG. 24a illustrates a microplate 2400 with cell incubation wells 2410and a region of machine readable information 2420. FIG. 24b illustratesa microplate 2430 with cell incubation wells 2440, a region of machinereadable information 2450 and a region for reagent deposits 2460. FIGS.24c-24d include depictions of regions of electrical interface elements.FIG. 24c illustrates a microplate 2470 with cell incubation wells 2472,a region of machine readable information 2474 and a region forelectrical interface 2478. FIG. 24d illustrates a microplate 2480 withcell incubation wells 2482, a region of machine readable information2484 and a region for electrical and optical interface 2488. FIG. 25illustrates electrical interface elements 2510 provided at each well2500 (and in some cases, reagent deposition site) and interface directlywith a cap 2520. Many other combinations are possible and areanticipated for various embodiments.

It is noted that the example arrangements depicted in FIGS. 24a-24dutilize circular shapes for indented wells and rectangular shapes of theregions of machine readable information. Further, the examplearrangements depicted in FIGS. 24b-24d and FIG. 25 employ rectangularshapes for reagent deposits, electrical interface elements, and opticalinterface elements. However, these shape choices are merely examples andother shapes (for example hexagonal, rhomboidal, trapezoidal, etc.) canbe used as found to be advantageous.

In some electrical sensing arrangements (such as has been describedearlier), the removable replaceable media element can further compriseadditional electrical elements including but not limited to electricalshielding, diodes, transistors, resistors, capacitors, inductors,ferrites, electronic circuitry, etc. as well as materials suitablyconductive, insulating, etc.

In some optical sensing arrangements (as will be described later), thewell and removable replaceable media element can further compriseoptical elements including but not limited to LEDs, photodiodes,phototransistors, etc. as well as materials suitably opaque,transparent, or translucent at specific wavelengths of electromagneticradiation, etc.

Machine Readable Information Provided by the Removable Replaceable MediaElement

Machine Readable Information provided by the removable replaceable caninclude data and/or algorithms and can take the physical form of printedoptical codes (such as printed optical bar codes, printed optical matrixcodes, printed holographic codes), printed magnetic code stripe, printedelectronic data memory, etc.).

In an embodiment, the machine readable information provided by theremovable replaceable media element is printed on the removablereplaceable media element.

In another embodiment, the machine readable information provided by theremovable replaceable media element is comprised by a machine readablemedium such as a separately manufactured label attached to the removablereplaceable media element by a melding, adhering, or other attachmentmethod or process.

In an embodiment, the machine readable information provided by theremovable replaceable media element comprises date informationassociated with the materials on the removable replaceable mediaelement.

In an embodiment, the machine readable information provided by theremovable replaceable media element comprises serial number information.

In an embodiment, the machine readable information provided by theremovable replaceable media element comprises information describing theincluded sensor configuration.

In an embodiment, the machine readable information provided by theremovable replaceable media element comprises information relating theincluded sensor operation.

In an embodiment, the machine readable information provided by theremovable replaceable media element comprises information specifyingparameters used by at least one algorithm.

In an embodiment, the machine readable information provided by theremovable replaceable media element comprises information specifying atleast one algorithm.

The interface and reading of the machine readable medium by the baseunit will be discussed with reference to FIGS. 30a -c.

Sensors Provided by the Microplate Removable Replaceable Media Element,Sensor Fabrication Via Printing, and Associated Printed Electronics

As described earlier and taught in Pending U.S. patent application Ser.No. 13/761,142, a wide variety of sensors, organic electronic sensors,and other printed devices relevant to the removable replaceable mediumelement aspects of the present application can be created through alayered implementation oriented in a manner suitable for printing.

FIGS. 26a-26b depict representations of example functional printedmethods that can be used, for example, to print the sensors on theremovable replaceable medium. For example, functional printing can beimplemented by rendering precision-controlled depositions of one or moretypes of fluid “inks” onto a surface. Here the “inks” can comprise oneor more of various types of electrical organic conductors, organicinsulators, organic semiconductors, reflective materials, antibodies,enzymes, colloidal substances, metamaterials, etc. Such “inks” can dry,polymerize, can be “cured,” etc., after deposition by employing varioustypes of drying, heating, evaporating-time pause, vacuum aspiration,photoactivation, and/or other processes. The “inks” can be applied inlayers to create layered structures comprising different materials andwell-defined interfaces between them. In some arrangements, the inks canbe blended in the printing (or other deposition) action. The inks mustpermit specified functions to properly occur (for example properimmobilization of biologically active materials, electrical conduction,charge carrier injection, etc,), have proper electrical, thermal, andmechanical characteristics, and be non-soluble in the fluids used tocarry the analyte.

Alternative Use of Silicon Semiconductors and Semiconductor Devices

Although printed semiconductor devices such as field effect transistorarrangements suitable for subsequent printing of a layer of selectivedetection material are expected to become straightforwardly fabricatedwith optimized materials at low cost with high levels of performance, atthe moment traditional silicon semiconductors typically offer higherperformance, for example due to carrier mobility issues in organicsemiconductors. Accordingly, the present application provides for theuse of silicon semiconductors and semiconductor devices.

As a first example, silicon-based semiconducting field effect transistorstructures with an exposed insulated gate (the insulated gatesubsequently metalized or not, depending on the design of the sensor atthe particular site) can be surface mounted on the removable replaceablemedium element, and printing of a layer of selective detection materialon the exposed gate (or metalized gate contact) can be performed. Inthis case, the removable replaceable medium element is not itself asilicon wafer, other sensor sites can be freely fabricated by printingof electrodes, organic field effect transistors, etc., and depositedmaterials such as reagents can be freely fabricated by printing or otherdeposition processes in other regions of the removable replaceablemedium element.

As a second example, such silicon-based semiconducting field effecttransistor structures can be surface mounted on every sensor site of theremovable replaceable medium element, and printing of a layer ofselective detection material on the exposed gate (or metalized gatecontact) can be performed. In this case, the removable replaceablemedium element is again not itself a silicon wafer, and depositedmaterials such as reagents can be freely fabricated by printing or otherdeposition processes in other regions of the removable replaceablemedium element.

As a third example, a plurality of such silicon-based semiconductingfield effect transistor structures can be rendered and sparselydistributed on a silicon wafer or portion of a silicon wafer that isattached to the removable replaceable medium element, and printing of alayer of selective detection material on the exposed gate (or metalizedgate contact) can be performed. In this case, the entire removablereplaceable medium element is not a silicon wafer, other sensor sitescan be freely fabricated by printing of electrodes, organic field effecttransistors, etc. in other regions of the removable replaceable mediumelement, and deposited materials such as reagents can be freelyfabricated by printing or other deposition processes in other regions ofthe removable replaceable medium element.

As a fourth example, a plurality of such silicon-based semiconductingfield effect transistor structures can be rendered and sparselydistributed on a silicon wafer or portion of a silicon wafer thatcomprises the entire substrate of the removable replaceable mediumelement, and printing of a layer of selective detection material on theexposed gate (or metalized gate contact) can be performed. In this case,the entire removable replaceable medium element is a silicon wafer, anddeposited materials such as reagents can be freely fabricated byprinting or other deposition processes in other regions of the removablereplaceable medium element.

As a fifth example, the above fourth example, additionally one or moreregions of electrodes are provided on the silicon wafer, either in asilicon wafer step or but subsequent printing of conductive material,and other sensor sites can be freely fabricated by printing of organicfield effect transistors, etc. in these “electrode only” regions of theremovable replaceable medium element, and deposited materials such asreagents can be freely fabricated by printing or other depositionprocesses in other regions of the removable replaceable medium element.

Other variations are anticipated and provided for by the presentapplication. For example, as described earlier, some electrochemicalsensors can benefit from direct connection of electrochemical electrodesto a field effect transistor and can be fabricated in ways combining theabove approach with those involving field effect transistors.

Electronics Interface Provided by the Removable Replaceable MediaElement

In some embodiments, electrical connections for one or more sensors orsensor components at a particular well can be routed to an electricalconnection region located at the associated well, surrounding the well,on at least one edge of the well, near at least one edge of the well,etc. on the removable replaceable media element, for electricalconnection through electrical contacts comprised by an associated cap(for example as depicted in FIG. 25), a group of caps, or otherarrangement, although many other variations are possible and providedfor by the present application.

In some embodiments, electrical connections for one or more sensors orsensor components can be routed to an electrical connection region inanother part of the removable replaceable media element. In someimplementations, at least one electrical connection for one or moresensors or sensor components is made to an electrical shieldingarrangement comprised by the removable replaceable media element. Insome implementations, at least one electrical connection for one or moresensors or sensor components is made to an electrical circuit (forexample, an amplifier, differential amplifier, current source,comparator, analog-to-digital converter, digital-to-analog converter,etc. the removable replaceable media element.

In some implementations, at least one electrical connection to anelectrical circuit (for example, an amplifier, differential amplifier,current source, comparator, analog-to-digital converter,digital-to-analog converter, etc. on the removable replaceable mediaelement is made to electrical connections on the removable replaceablemedia element arranged to electrically connect with electricalconnections at the associated site, surrounding the site, on at leastone edge of the site, near at least one edge of the site, etc. on theremovable replaceable media element.

As described earlier, FIGS. 24c-24d include depictions of centralizedregions comprising electrical interface elements. Alternatively, or inaddition, electrical interface elements can be provided at each well(and in some cases, reagent deposition site).

Optical Interface Provided by the Removable Replaceable Media Element

As an alternative to a plurality of electrical contacts for carryingsensor signals from the Microplate Removable Replaceable Media Element,electronics provided within the Microplate Removable Replaceable MediaElement can be used to convert the electrical signals into opticalsignals. Using this approach, electrical connections can be used forpowering the Microplate Removable Replaceable Media Element whileoptical signals can be used for carrying a rich density of sensorsignals. FIG. 24d includes a depiction of centralized regions comprisingoptical interface elements.

Various embodiments can multiplex pluralities of sensor signals into oneor at least a considerably smaller number of multiplexed signals ofdigital or analog format.

Base Unit and its Interfacing Removable Replaceable Media Element

Various embodiments provide for interactions between the removablereplaceable media element (for example, removable replaceable mediaelement 2210 of FIG. 22a , element 2292 of FIG. 22b , and the removablereplaceable media elements 2330, 2370 and 2395 of FIGS. 23a-23c ) andthe base unit (for example, base unit 2250 of FIG. 22a , base unit 2294of FIG. 22b , and base units 2300, 2335 and 2375 of FIGS. 23a-23c ).

Regarding sensors or sensor components on the removable replaceablemedia element that comprise electrical connections, each “cap” can beconfigured to cover an associated region of a removable replaceablemedia element that comprises one or more, the cap can compriseassociated electrical connections for making electrical contact withcorresponding electrical connection on the removable replaceable mediaelement. The electrical connections for one or more sensors or sensorcomponents at a particular well can be routed to an electricalconnection region located at the associated well, surrounding the well,on at least one edge of the well, near at least one edge of the well,etc. on the removable replaceable media element, for electricalconnection through electrical contacts comprised by an associated cap, agroup of caps, or other arrangement.

In some electrical sensing arrangements (for example as taught inPending U.S. patent application Ser. No. 13/761,142), the cap canfurther comprise electrical elements including but not limited toelectrical shielding, diodes, transistors, resistors, capacitors,inductors, ferrites, LEDs, photodiodes, phototransistors, electroniccircuitry, etc. as well as materials suitably conductive, insulating,etc.

In some optical sensing arrangements (for example as taught in PendingU.S. patent application Ser. No. 13/761,142), the cap can furthercomprise optical elements including but not limited to LEDs,photodiodes, phototransistors, etc. as well as materials suitablyopaque, transparent, or translucent at specific wavelengths ofelectromagnetic radiation, etc.

FIG. 27a depicts an example representation showing “cap” 2700 (withoutillustrating fluidic ports, electrical connections, mechanical support,etc.) interfacing with a site or area 2710 within a removablereplaceable media element. In some embodiments cap 2700 can be used tointerface individual wells or groups of wells. In some embodiments cap2700 can be nested in various hierarchical arrangements.

A cap can also be used with a removable replaceable media element withsensors or sensor components. FIG. 27b depicts an example representationof cap 2720 that covers a site area 2730 within a removable replaceableelement that comprises a sensor 2740 (here abstractly represented as abold rectangular solid).

A cap can also be used with a removable replaceable media element withreagents. FIG. 27c depicts an example representation wherein the cap2750 covers a site or area 2760 within a removable replaceable mediaelement that comprises a printed deposition 2770 comprising one or morereagents or other materials (for example, gas-generating, soap,emulsifier, disinfectant, etc.), for example, in the form of a solublesolid or gel comprising a soluble reagent or other type of material. Thedeposition can be functionally structured so as to provide awell-defined dissolution process in the fluid-exchange environmentwithin the cap that does not result in problems such as sedimentation,loose fragments that could clog fluidic ports, clog fluidic valves,provide uncontrolled variations in concentration, or affect sensoroperation. For example, the soluble solid or gel can comprise a polymerlattice, zeolite-like structure, etc. Depending upon the approach taken,the soluble solid or gel can comprise a soluble solid reagent, a mixableor soluble liquid reagent previously entrapped (macroscopically ormicroscopically) within the soluble solid or gel structure, and even agas (for example entrapped within the structure or resulting from achemical or enzymic reaction, etc.).

FIG. 28 depicts an example representation of a removable replaceablemedia element 2800 wherein cap 2810 covers a site or area that comprisesa printed reagent or material deposition 2830, such as solid or gel, andcap 2820 covers a site or area that comprises a printed reagent ormaterial deposition 2850, such as solid or gel. Caps 2810 and 2820 areprovided with respective input fluidic ports for accepting fluids in andrespective output fluidic ports for carrying dissolved reagent outward.The arrangement can also be used for catalytic regents operating onincoming liquids or gasses. Although untapered square-opening (cap 2820)and round-opening caps (cap 2810) are depicted in FIG. 28, other capshapes can be used.

Controllable Valves

The various embodiments of the present application can incorporatefluidics at various scales of physical size, ranging from those that usesmall-scale convention and fittings, controllable valves, pumps, andfluidic conduit manufacturing techniques to microfluidic scalesinvolving transport of nanoliter volumes of materials. The value of thesystem would be expected to increase with increasing degrees ofminiaturization as fewer sample, supplies, and power are required, fielduse is better facilitated, etc. As the scale of physical size decreases,the implementation of valves and pumps becomes less conventional and newemerging approaches and techniques will be used.

Further, these less conventional approaches and techniques are expectedto continue to evolve. Many of these employ either elastomeric materialsthat response to applied pressure forces or complex polymers that changetheir physical properties responsive to electricity or heat. These andother know and as yet unknown approaches and techniques are expected tocontinue to evolve, emerge, and compete. However, as seen in the listabove, there are many approaches that ultimately can be controlled byelectrical current and/or voltage processes, making theme suitable forcontrol by a microprocessor, other computation system, and/or digitallogic circuitry.

Some examples of controllable valves suitable for microfluidics systemsinclude but are not limited to those operated by:

-   -   Pneumatic or hydraulic stimulus (as for example, can be induced        by larger scale apparatus driven by and controlled by electrical        voltage or current)    -   Thermal processes (induced by electrical resistance or        electrically produced infrared radiation)    -   Piezoelectric actuation (as for example, can be driven by        electrical voltage or current)    -   Magnetic fields (as for example, can be induced by electrical        current)    -   Torque or other mechanical actuation (as for example, can be        induced by larger scale apparatus driven by and controlled by        electrical voltage or current)

As one specific example, piezoelectric actuators can be used tomanipulate elastomeric materials, either by direct mechanical contact ofthrough intermediate pneumatic or hydraulic transfer. As anotherspecific example, an electrically controlled microvalve leveraging largevolumetric phase-change, for example as occurring in polyethylene glycolpolymers (PEG), are thermally controlled using thin film resistiveelements patterned using standard microfabrication methods, for exampleas taught in G. Kaigala, V. Hoang, C. Backhouse, “ElectricallyControlled Microvalves to Integrate Microchip Polymerase Chain Reactionand Capillary Electrophoresis,” Lab on A Chip, 2008, Vol. 8, No. 7, pp.1071-1078 (whose authors indicate can readily scale down in size andrequire only electrical connection).

It is noted that thin films, elastomeric materials, and polymers can,through various processes and preparation, be functionally printed.Additionally, various practical aspects of the fabrication and operationof microfluidic valves based on elastomeric materials can be found, forexample, in B. Mosadegh, Design and Fabrication of MicrofluidicIntegrated Circuits Using Normally Closed Elastomeric Valves, UMIDissertation Publishing, 2010, ISBN 9781244570306.

Pumps

As will be seen, in many approaches supported by the present applicationanalyte propagates through one or more serial chains of processing andsensing regions, and if there is more than one serial chain at least onefan-out stage is involved; these arranged in a manner that could beadequately managed with a single pump and the operation of valves tocontrol where flow is active or blocked. Accordingly, a single or smallnumber of pumps arranged for transport of small amounts of fluid buthaving a comparatively considerably larger overall physical size (forexample, a miniature motor-driven, solenoid driven, orpiezoelectric-driven diaphragm pump, a miniature motor-driven,solenoid-driven, or piezoelectric-driven peristaltic pump, a miniaturemotor driven, solenoid-driven, or piezoelectric-driven syringe pump,etc.).

As to microfluidic pumps, as with microfluidic valves there are manyapproaches that ultimately can be controlled by electrical currentand/or voltage processes to control or induce a mechanical actuation. Inmany cases the same types of mechanical actuation used to operate avalve can be used to operate a diaphragm pump, actuate a steppingmechanism for a syringe pump, and arranged in a sequenced ensemble ordrive a rocker arrangement to create a peristaltic pump. Many examplesof these can be found in the literature, and it is expected that theseand other know and as yet unknown approaches and techniques are expectedto continue to evolve, emerge, and compete. However, as seen in the listabove, there are many approaches that ultimately can be controlled byelectrical current and/or voltage processes, making theme suitable forcontrol by a microprocessor, other computation system, and/or digitallogic circuitry. Further as to microfluidic pumps, much attention in themicrofluidics literature has been directed to electro-osmotic transport.Although the present application provides for the use of electro-osmotictransport where applicable or advantageous, it is noted that theelectric fields and introduced voltage potentials involved can affectbiomolecules, cells, suspensions, etc, can introduce unwanted orunmanageable electrochemical effects, and can interfere with theintended operation of many types of sensing technologies and processes,Accordingly, in some embodiments electro-osmotic transport is employedwhere applicable or advantageous to transport materials (or a somewhatrestricted class of materials) between fluidic locations but isnonoperational when sensing that could be affected by voltages, current,and fields associated with electro-osmotic operation. The presentapplication in a similar manner provides for the use of other similarpump techniques, for example as taught in S. Chang, E. Beaumont, D.Petsev, O. Velev, “Remotely Powered Distributed Microfluidic Pumps andMixers Based on Miniature Diodes,” Lab on a Chip, 2008, Vol. 8, pp.117-124.

Organization and Handling of Fluidic, Gas Exchange, and Other MaterialFlows

With valves and pumps applicable to the present application nowdiscussed, attention is now directed to interfacing the sites on theremovable replaceable media element to fluidics within or intermediateto the base unit, as well as electronics and optics within orintermediate to the base unit, and associated interconnection. Attentionis first directed to fluidics interfacing, fluidic control, and fluidicsinterfacing.

As an example, fluidic and gas exchange interconnections can be providedby controllable microfluidic bus technologies such as that taught inU.S. Pat. No. 8,032,258 and pending U.S. patent application Ser. Nos.13/251,286 and 13/251,288). Fluidic interconnections among caps andassociated sites can be supplemented with additional controllablefluidic paths. In some implementations fluidic interconnections carryfluids, gases, solvents, cleaning fluids and/or clearing gases (such asthat taught in pending U.S. patent application Ser. Nos. 11/946,678 and13/314,170) and/or can connect to controllable microfluidic bus (such asthat taught in pending U.S. Pat. No. 8,032,258 and pending U.S. patentapplication Ser. Nos. 13/251,286 and 13/251,288). The fluidicsarrangements described thus far understood to extend to cover materialsand situations such as suspensions (for example comprising cells), gasesdissolved liquids, materials at thermodynamic critical points (such asvapors and gases including vapors), slurries, gases comprisingparticulates or colloids, emulsions in various stages (flocculation,creaming, coalescence, Ostwald ripening, etc.), micelles, etc. as wellas combinations of these. In various aspects of the present application,the fluidics arrangements are arranged to interface to wells andreagent, depositions on the removable replaceable media element.

FIG. 29 depicts an example arrangement wherein caps 2910 areinterconnected with fluidics and gas exchange arrangements 2920 tointerface with wells and reagent deposits on the removable replaceablemedia element 2900.

Fluid Reservoir

In an embodiment, at least one fluid reservoir is provided in the baseunit.

In an embodiment, the fluid reservoir is removable.

In an embodiment, the fluid reservoir is built into the removablereplaceable medium element, for example within or beneath thesensor-level substrate removable replaceable medium element.

In an embodiment, at least one disposal reservoir is provided in thebase unit.

In an embodiment, that disposal reservoir is removable.

In an embodiment, the disposal reservoir is built into the removablereplaceable medium element, for example within or beneath thesensor-level substrate removable replaceable medium element.

In an embodiment, the fluid reservoir and disposal reservoir are in aunitary removable configuration.

Machine-Readable Information Aspects of the Base Unit

As described earlier, the machine readable information provided by theremovable replaceable can include data and/or algorithms and can takethe physical form of printed optical codes (such as printed optical barcodes, printed optical matrix codes, printed holographic codes), printedmagnetic code stripe, printed electronic data memory, etc.).

As an example, FIGS. 30a-30c depict representations of examples of howoptical ROM printed on the removable replaceable media can be read bythe base unit. FIG. 30a depicts an example linear (1-dimensional)optical “bar” code 3010 that can be printed on instances of theremovable replaceable medium and a “reading array” 3020 comprising forexample a 1-Dimensional Photodiode Array, 1-Dimensional LED Array,1-Dimensional CCD Array, etc. located in the base unit and configured tolie effectively optically adjacent to the optical bar code printed onthe removable replaceable media. No mechanical scanning is needed withthis approach. (As described later, LEDs can operate aswavelength-selective photodiodes.) The barcode 3010 can be lit byvarious arrangements, including back lighting, frontal light, viaselected LEDs in an LED array, etc. Translational-displacement of theoptical bar code 3010 with respect to the “reading array” 3020 (arisingfrom minor variations in removable replaceable media positioning withrespect to the base unit) can be readily handled in software on amicroprocessor or other processor chip comprised by the base unit. Ifthe “reading array” 3020 comprises LEDs with a number of distinguishabledifferent emission wavelengths, the LED array can be used to implementwavelength division multiplexing arrangements, allowing use of multiplecolored inks used in the printing of the optical bar code 3010 toincrease the information density on the optical bar code 3010.

FIG. 30b depicts an example elongated rectangular 2-dimensional optical“matrix” code 3030 that can be printed on instances of the removablereplaceable medium and a “reading array” 3040 comprising for example anelongated rectangular 2-Dimensional Photodiode Array, 2-Dimensional LEDArray, 2-Dimensional CCD Array, etc. located in the base unit andconfigured to lie effectively optically adjacent to the optical matrixcode printed on the removable replaceable media. No mechanical scanningis needed with this approach. The matrix code 3030 can be lit by variousarrangements, including back lighting, frontal light, lighting by LEDsin an LED array, etc. Translational displacement of the matrix code 3030with respect to the “reading array” 3040 (arising from minor variationsin removable replaceable media positioning with respect to the baseunit) can be readily handled in software on a microprocessor or otherprocessor chip comprised by the base unit. If the “reading array” 3040comprises LEDs with a number of distinguishable different emissionwavelengths, the LED array can be used to implement wavelength divisionmultiplexing arrangements, allowing use of multiple colored inks used inthe printing of the optical matrix code 3030 to increase the informationdensity on the optical matrix code 3030.

FIG. 30c depicts an example non-elongated rectangular 2-dimensionaloptical “matrix” code 3050 that can be printed on instances of theremovable replaceable medium and a “reading array” 3060 comprising forexample a non-elongated rectangular 2-Dimensional Photodiode Array,2-Dimensional LED Array, 2-Dimensional CCD Array, etc. located in thebase unit and configured to lie effectively optically adjacent to the2-d optical matrix code 3050 printed on the removable replaceable media.No mechanical scanning is needed with this approach. The 2-d opticalmatrix code 3050 can be lit by various arrangements, including backlighting, frontal light, lighting by LEDs in an LED array, etc.Translational displacement of the 2-d optical matrix code 3050 withrespect to the “reading array” 3060 (arising from minor variations inremovable replaceable media positioning with respect to the base unit)can be readily handled in software on a microprocessor or otherprocessor chip comprised by the base unit. If the “reading array” 3060comprises LEDs with a number of distinguishable different emissionwavelengths, the LED array can be used to implement wavelength divisionmultiplexing arrangements, allowing use of multiple colored inks used inthe printing of the optical matrix code to increase the informationdensity on the optical matrix code.

Alternatively, other arrangements for optical ROM, electronic ROM (forexample, implemented with printed electronics), magnetic ROM, etc.) canalso be used.

In an embodiment, compressed specification languages and procedurallanguages can be used to minimize the number of characters stored on theROM.

Separable Interfacing Module

In many implementations and usage scenarios it can be advantageous toimplement at least the fluidics arrangements in a separable interfacingmodule. In some embodiments the interfacing module can be comprised bythe base unit in either a fixed or replaceable arrangement. In otherembodiments the interfacing module can be comprised by the removablereplaceable media element in either a fixed or replaceable arrangement.In yet other embodiments the interfacing module can be configured to beinserted into either (at the choice of user or manufacturerproduct-design) the base unit or attached to the removable replaceablemedia element in either a fixed or replaceable arrangement.

In various embodiments, the interfacing module can additionally compriseone or more of various additional components including but not limitedto electronic circuitry, valves or portions of valves, optical elements,electro-optical elements, mechanical actuators, pumps, reservoirs,

microprocessors, additional sensors, etc.

Further as to the “open architecture” provisions of the presentapplication involving removable replaceable interface module, FIG. 31depicts an example adaptation of the example architectural arrangementillustrated in FIG. 22b wherein a removable replaceable interface module3110 is provided to interface to the microfluidics 3120 and computinginfrastructure 3130. Other architectural arrangements are of coursepossible, anticipated, and provided for by the present application.

In various embodiments, the interface module 3110 can be fabricated inpart or in whole by functional printing. In various embodiments, theinterfacing module can be fabricated in part or in whole by injectionmolding. In various embodiments, the interface module 3110 can befabricated in part or in whole by casting.

When the interface module 3110 is employed as a removable component foruse in the base unit, such an arrangement allows for simplifiedmaintenance, performance upgrades, density upgrades, feature upgrades,means of contamination control within the base unit, etc. When theinterface module 3110 is employed as an attached or user-attachablecomponent to the removable replaceable media element, such anarrangement allows for containment of contamination, simplified usage,performance customizations, density customizations, featurecustomizations, etc.

FIG. 32 depicts a variation on the example arrangement of FIG. 29wherein at least the fluidics arrangements are comprised in aninterfacing module. FIG. 32 illustrates an interfacing module 3200comprising fluidics. FIG. 33 depicts an example wherein the interfacingmodule 3300 (e.g., interfacing module 3200) can be configured to beinserted into either (e.g., at the choice of user or manufacturerproduct-design) the base unit or attached to the removable replaceablemedia element 3310 in either a fixed or replaceable arrangement.

Software

FIG. 34 depicts a representation of a system 3400 according to certainembodiments. System 3400 includes applications software 3410, which caninclude, for example, configuration data, assignment data, data used byalgorithms, test algorithms, analysis algorithms, etc. A well (andreagent) configuration table 3420 that can be used to specify, amongother things, what mode each well is to operate in. System 3400 includesfluidic configuration data 3430 that can be used to configure fluidicelements 3432, such as valves (and which can also include pumps). System3400 includes electronic configuration data 3440 that used to configureelectronic elements 3442, such as sensor interface electronics, logicgates, routing of sensor signals to A/D converters, mixed signalintegrated circuits, digital processors, microprocessors, etc. System3400 includes one or more algorithms 3450, such as test algorithms 3452(e.g., standard or special operation of sensors and fluidics, etc.), aswell as analysis algorithms 3454, e.g., used to produce the statisticalanalysis of the information provided by the sensors. Algorithms 3450 arestored in memory 3460 and executed by microprocessor 3470.

User Interface

FIG. 35 depicts a representation of an overall example user experiencescenario using an example implementation of the technology. The depictedsteps, features, elements, event sequence, clustering, and flow aremerely illustrative and are in no way limiting.

Example Applications Enabled by Embodiments

This section provides an example experiment showcasing exampleapplications that are enabled by various embodiments described earlierand which otherwise, if they can even be performed at all, can beperformed only with great laboratory skill, high experiment-corruptionrisk, and with poor efficiently

As an overview, FIG. 36 depicts a representation of the strategy of anexample experiment facilitated by various embodiments. FIG. 37 depicts aflow chart of an example experiment leveraging various aspects ofvarious embodiments. FIG. 38 depicts a representation of how measurementdata obtained from parallel hypoxia and normoxia conditions arecompared. FIG. 39 depicts a representation of how NO donor materialprofiles can be studied. FIG. 40 depicts a representation ofexperimental processes for studying effect of NO on cell survival.

As is known, Nitric Oxide (NO) acts as double-edged sword, which inducesand prevents cell death, depending on various factors. The mechanism forthe NO regulation of cells is not fully understood. Various embodimentssupport the design and rapid conducting of intricate comparativeexperiment and therapy design methods leveraging viable hypothesissupported by current research findings. In comparison, conventional cellincubators cannot provide the variability and degrees of controlnecessary.

Various embodiments can be used to effectively determine the effects ofNO by controlling the majority parameters such as the steady-stateconcentration of NO, the duration of NO exposure, and the local level ofoxygen. Such experiment designs facilitated by various embodiments arestructured to improve the poor understanding of NO regulation of cells,and further can be directly adapted for therapy design.

Embodiments of the present application directs these experiment designand therapy design methods to the study and treatment of cancer. In theembodiments discussed herein, although cancer cells are discussed, othertypes of cells can also be used. In an application, the effects ofexogenous NO on inducible Nitric Oxide Synthase (iNOS) expression andsignal transduction in ovarian cancer is applied to drug discovery andtherapy design for ovarian and other cancers. An example framework ofsuch experiments is provided in pending U.S. patent application Ser. No.13/155,370 by one of the inventors, and the example framework providedtherein is considered here as an example for showcasing the valueprovided by various embodiments over the value available from currentand previous cell incubator technologies.

In the context of the example experimental framework provided in pendingU.S. patent application Ser. No. 13/155,370, various embodiments supportthe design and rapid conducting of intricate comparative experiments forthe study of Nitric Oxide (NO) in the control of cell death for use incancer therapies, the method comprising:

-   -   Establishing that long term exposure of cells to low        concentration of exogenous nitric oxide causes a negative        feedback mechanism to induce down regulation of inducible Nitric        Oxide Synthase (iNOS) by choosing a first NO donor that can        produce a relatively low level of NO concentration over a        relatively long NO-production half-life,    -   Applying that NO donor to a cell culture media at selected time        points, measuring the levels of iNOS concentration and NO        concentration to produce first measurements, and recording first        data corresponding to the first measurements;    -   Establishing that high concentration of exogenous NO does not        affect to the expression of iNOS but instead will cause        apoptosis of cells by choosing a second NO donor that can        produce a relatively high level of NO concentration over a        relatively short NO-production half-life, applying that NO donor        to a cell culture media at selected time points,    -   Measuring NO concentrations to produce second measurements, and        recording second data corresponding to the second measurements;        and establishing that for a given level of NO concentration, the        expression of iNOS down-regulates more in hypoxic conditions        than in normal oxygen conditions by choosing a third NO donor        that generates a relatively low concentration of NO,    -   Applying that NO donor to a cell culture media at selected time        points, measuring the levels of iNOS and NO concentration to        produce third measurements,    -   Recording third data corresponding to the third measurements,        and comparing the iNOS response between normal and hypoxic        environments, wherein at least one of the first data, second        data, and third data are used to create a cancer therapy that        manipulates exogenous NO in a hypoxic area of cancer cells.

Various embodiments support methods for designing a cancer therapy bymanipulating the concentration and duration of Nitric Oxide (NO) for thecontrol of cell death, the method comprising:

-   -   Using a first NO donor to produce a relatively low level of NO        concentration over a relatively long NO-production half-life,    -   Applying that NO donor to a cell culture media at selected time        points, measuring the levels of inducible Nitric Oxide Synthase        (iNOS) and NO concentration to produce first measurements, and        recording first data corresponding to the first measurements;    -   Using a second NO donor to produce a relatively high level of NO        concentration over a relatively short NO-production half-life,        applying that NO donor to a cell culture media at selected time        points, measuring the levels of iNOS and NO concentration to        produce second measurements, and recording second data        corresponding to the second measurements; and    -   Using a third NO donor to produce a relatively low concentration        of NO, applying that NO donor to a cell culture media at        selected time points, measuring the levels of iNOS and NO        concentration to produce third measurements, recording third        data corresponding to the third measurements, and comparing the        iNOS response between normal and hypoxic environments, wherein        at least one of the first data, second data, and third data are        used to create a cancer therapy that manipulates exogenous NO in        a hypoxic area of cancer cells.

Such a cancer therapy comprises an agent manipulates exogenous NO in thehypoxic area of cancer cells.

Various embodiments support the design methods for a cancer therapy, themethod comprising:

-   -   Using experimental measurements to find ranges of Nitric Oxide        (NO) concentrations and exposure durations which cause apoptosis        of cancer cells residing in a hypoxic environment;    -   Identifying an agent that can produce said ranges of Nitric        Oxide (NO) concentrations and exposure durations, and    -   Deploying the agent into a hypoxic area comprised by cancer        cells,        wherein the agent manipulates the concentration and duration of        Nitric Oxide (NO) within said ranges to invoke apoptosis of the        cancer cells.

Much circumstantial evidence suggests a hypothesis that low NOconcentration causes the progression of cancer cells, but high NOconcentration induces cancer cells apoptosis. However, no evidence hasisolated the factor or process that controls the negative feedback of NOto iNOS expression. Candidates for such factor or process can be arguedto prominently include the local molecular concentration and temporalduration of NO exposure events.

The NO mechanism is not clearly elucidated as to the conditions andprocesses wherein NO induces cancer cell progression or wherein NOpromotes cell death. Accordingly, experiment designers usingconventional cell incubators are faced with the prospect of trying tofind a needle in haystack in trying to answer how NO regulation on tumorcells.

Due to in fact that iNOS plays a role in cancer progression andmetastasis [12] and one of all pathways that regulate cancer progressionis negative feedback of NO to iNOS, it can be hypothesized that longterm exposure of cancer cells to low concentration of exogenous NO mightreduce iNOS expression by negative feedback effect. In contrast, highconcentration and short term of NO exposure might not regulate iNOSexpression and retards the survival of cancer cells. Another hypothesisthat can be made is that low concentration of NO causes down-regulationof iNOS under hypoxia more than in normoxia. Since high level of oxygenin the environment, normoxic condition increases metabolism of NO.Understanding mechanisms controlling the balance between the NO relatedprotective and destructive action can therefore be potentially be acrucial modification of the regimen of exogenous NO for alternativetumor therapy.

Quantitative Measurement of Nitric Oxide Via Spectroscopic Method NO Kit

NO contained in a sample is metabolized to nitrate and nitrite, and thennitrate is converted to nitrite by nitrate reductase. The nitrite willreact with Griess reagent, an azo compound, which causes the color toturn purple which can thus be detected by spectrophotometer FIG. 41depicts the principle of operation of such an NO quantitative kit. Inthis manner, the concentration NO resulting from the NO metabolite(nitrite) can be determined. However, NADPH, a crucial cofactor fornitrate reductase, decreases the sensitivity of this method byinterfering the Griess reaction. To increase the sensitivity, lactatedehydrogenase (LDH) can be added to the step prior to Griess reaction soas to address the NADPH scavenged cofactor.

Electrochemical NO Detection

NO is a highly reactive molecule and can be oxidized to be otherreactive oxygen species such as superoxide anion (O₂ ⁻ and the hydroxylradical (⁻OH), peroxynitrite (ONOO—). Electron transfer between the“active” electrode and the molecule causes an electrical current. Theelectrical current, redox current, is proportional to the concentrationof the molecules. Therefore the redox current is amplified and then itcan indicate the concentration of NO in Amperometric analysis. Shibukiinvented the first NO— sensitive probe in 1990 [4]. The improvement ofthe NO probe has focused on the sensitivity, selectivity andreliability. However the main problem the electron exchange with thechemical species is very slow; consequently it induces a low responsetime and poor sensitivity. There are two solutions for solving thedifficulty. First one is increasing the applied potential—gaining theenergy of the electrons. In fact, this method reduces a signal to noiseratio and leads to the loss of selectivity, even though it increases theresponse rate of electrons. Second strategy is using a catalyst toreduce the activation energy. This strategy minimizes the energy gapbetween the working electrode and the related chemical; hence, itimproves the electron transfer and increases the signal to noise ratioand the selectivity [5,6]. From the study of Brovkovych et all, 1990,the catalyst using in reducing the energy gap is porphyrin. They developphophyrin microsensors based on coated carbon fibre. Phorphyrin coatingdiminished the potential to 0.7 V and improves the responsesignificantly [7].

The electrochemical NO detection's systems are commercial available,such as, Amino—700 from the Innovative Instruments, Inc. and ISO-NOsystem from WPI Inc. These systems embody NO permeable membrane toreduce interference from other oxidized molecules. The electricalcurrent responds to changes of temperature [8]. Despite this NOapplication, the electrochemical detection is also used in othermolecules, such as, oxygen and oxygen derivatives, carbon dioxide,hydrogen sulfide glucose, and ATP.

An example of measuring NO concentration by using electrochemical NOdetection is Jean A. Cardinale's experiment which measured Nitric oxideconcentrations using an Apollo 4000 Free Radical Analyzer with ISO NOPsensors (World Precision Instruments, Sarasota, Fla.). To determine theconcentrations of NO, this study created a standard curve for conversionof pA (the electric current density unit) to nM (concentration) bytitrating of concentration of a standard potassium nitrite solution to asolution of 0.1 M KI (potassium iodide) in 0.1 M H₂SO₄ (sulfuric acid).The reaction was performed at 37° C. [21].

Fluorescence Indicator of NO

Many substances such as diaminofluoresceins (DAF-2) react with NO andform a fluorescent complex. The reaction in the presence of oxygencreates a bright green fluorescent triazolo derivative. In addition,other NO indicators such as diaminorhodamine (DaR), ordiaminoantraquinone (DAQ or DAA) chemically react with NO irreversibly.From the study of glial cell culture, the signal of DAF-2—NO complex isextremely small, in the range of 8 nM [9]. In addition, the resultsmodulation of neurotransmission requires NO concentration as 0.5-10 nM[8].

Fluorescence Resonance Energy Transfer (FRET) Detection of NO

Pairs of fluorophores act as donor of photons and the other as theiracceptor. The FRET indicator such as Cyan and yellow mutants of thegreen fluorescence proteins (GFP) were used to measure the concentrationof biological active molecules. A deep blue light from the indicators isused to excite the cyan fluorescence protein to emit blue photons.Consequently, the yellow fluorescence protein traps the blue photon. Asa result, the ratio between the fluorescence signals, initiated by thecyan and yellow proteins depends on the configuration of the moleculeand the distances between them; and may be used as an index of FRETintensity. The first discovery of an NO-sensitive FRET-based probe wasmade by Honda and his colleagues [31]. The study explained that cGMPdependent protein kinase (PKG) acts as NO sensor between two GFPmutants. In fact, PKG is sensitive to cGMP, which depends on theactivation of sGC. Therefore, this phenomenon is reported as NO—mediatedeffects.

Measurement of NO Effects in Retro-Dialysis Experiments

Micro-dialysis experiments are able to determine NO metabolites.However, the limitation of the experiments is slow [10].

Detection of NO by Electron-Paramagnetic Resonance (EPR)

EPR Spectroscopy is responsive to NO trapping invoked by a stablecomplex, and can be used to indicate the NO trapping location. In themeantime, it is able to estimates the concentration of NO at differentlocations and is accordingly useful to identify the release of NO [8].Further, because most stable molecules have all electrons paired,ordinary chemical solvents and matrices do not give rise to EPR spectra,simplifying and significantly improving the noise environment in themeasurement.

MTT Colorimetric Cell Assay for Measurement of Cellular Proliferation

To study the cellular proliferation of cancer cell lines, each type ofcancer cell line can be incubated with selective NO donor at theconcentration that will release a steady optimal concentration. Celllines can be sampling at each time point for studying cellularproliferation by using MTT reagent. The inhibition rate (RT) of theselective NO donor can be calculated from the absorbance reading, whichcan be taken using a microtiter plate reader, according to the followingformula:

% IR=(1−OD_(experiment)/OD_(control))×100

The results of such a test show when the culture cells have the highest% IR (that is, the time point at which NO will start to inhibit cellularproliferation) and what concentration will cause the maximum of % IRassociated with the concentration of cell death. In addition, theresults also identify the range of NO concentration and time exposurefor which the cells survive.

Microfluidic Cell Assay

Immunoassays is a technique that detects fluorescent signal from theinteraction between antigen—antibody. This assay can be utilized to theproteins and molecules' detection. Generally, the assay is performed in96-well microtiter plates and requires multiple interactions and washingsteps, and a long incubation period. However, to perform an assay, largevolume of expensive reagents, time consuming and inconvenience areproblematic. Therefore, an embodiment of microfluidic technology such aslab-on-a-chip and micro total analysis system has been developed toreplace the immunoassay's problems.

Due to in fact that NO has a short half-life, unstable, and we determinethe response of iNOS to the concentration of NO during the period oftime, NO quantitation measurement is one part of the crucial experimentsof this research. NO releasing was measured by observing the converse ofNO to nitrite and nitrate in the tissue culture media and this method isalso available in the commercial as NO quantitation kit.

EXPERIMENT DESIGN

To test the above hypothesis, one can for example pursue the followingexample specific aims:

Example Aim 1

Determine if level of NO and time of NO exposure regulate the expressionof iNOS and apoptosis.

An example working hypotheses is that long term of cells exposing to lowconcentration of exogenous nitric oxide causes a negative feedbackmechanism inducing down regulation of iNOS and promotes tumorprogression. As is well known, low NO concentration reduces apoptosis.Thus, low concentration of exogenous NO causes down regulation of iNOSresulting in a reduced ability to produce NO from iNOS. The lower levelof NO resulting from down-regulated iNOS will prolong the lifetime ofcancer cells. FIG. 42 summarized the relationship between NO exposureconcentration and duration (control variables in the experiment) andcell survival time.

Due to in fact that NO levels are needed to regulate in low ranges, theexperiment of this study requires in process measurement of NOconcentrations during cell culture. Hence, an incubator embodimentprovides the convenience, accuracy, and precision of NO concentrationmonitoring during cell incubating. For instance, NO donor is added toeach cell culture well. The sensors beneath the cell culture wellsmeasure the levels of NO. In the meantime, NO donor is added to thecells, when NO levels are dropped out of range of desire (NO levels aredetected by the sensor of incubator) to maintain steady state of lowconcentration of NO. Finally, the cell responses including iNOSexpression are able to determine after incubation time and concentrationof NO are completed.

In addition, cellular survival can be assessed using a MTT assaydescribed above. It can be predicted that the MTT assay will demonstratea low value of the inhibition rate (% IR) resulting from a long periodof low level of NO exposure.

Therefore, to determine the cell responses to NO concentration such asMTT assay, the fluidic plate of incubator embodiment allows theoperation of the reaction occurs while the cells are incubated in theregulated condition. Reactant fluids are moved and mixed in the fluidicplate in regulated environment. In addition, optical embodiment measuresabsorbance of the final product of the reaction.

Most reports have shown that high concentrations of NO have adestructive effect. In despite of low concentration, it can be predictedthat concentrations of NO high enough to cause cell apoptosis will notaffect to the negative feedback mechanism of iNOS. Therefore, it can bepredicted that the expression of iNOS (for example, as measured byWestern Blot or other methods) will not change after the cells areexposed to high level of exogenous NO, however, this same highconcentration will give high value of % RT from MTT assay.

Due to in fact that NO levels are needed to regulate in high ranges, theexperiment of this study requires in-process measurement of NOconcentrations during cell culture. Hence, embodiments of the presentapplication provide the convenience, accuracy, and precision of NOconcentration monitoring during cell incubating. For instance, NO donoris added to each cell culture well. The sensors beneath the cell culturewells measure the levels of NO. In the meantime, NO donor is added tothe cells, such that NO levels drop out of range of desire (NO levelsare detected by the sensors of incubator) to maintain steady state oflow concentration of NO. Finally, the cell responses including MTT assayand iNOS expression are able to be determined after incubation time andconcentration of NO are completed.

In addition, cellular survival can be assessed using a MTT assaydescribed above. It can be predicted that the MTT assay will demonstratea low value of the inhibition rate (% IR) resulting from a long periodof low level of NO exposure.

Most reports have shown that high concentrations of NO have adestructive effect. In despite of low concentration, it can be predictedthat concentrations of NO high enough to cause cell apoptosis will notaffect to the negative feedback mechanism of iNOS. Therefore, it can bepredicted that the expression of iNOS (for example, as measured byWestern Blot or other methods) will not change after the cells areexposed to high level of exogenous NO, however, this same highconcentration will give high value of % RT from MTT assay.

Example Aim 2

A High Level of Oxygen in the Cellular Environment Causes a normoxiccondition which increases metabolic use of NO. However, low levels ofoxygen cause a hypoxic condition that will maintain NO in the cellularenvironment because hypoxic conditions retards NO degradation.Therefore, it can be predicted that the duration of NO exposure underhypoxia will be longer than time of NO exposure under normoxia.Increasing time of NO exposure will cause down-regulation of iNOS bynegative feedback effect. One can observe iNOS expression, for example,using a Western Blot method.

Such work facilitated by various embodiments demonstrates how NO playsrole in apoptosis mechanism in cell by presenting the expression of iNOSas negative feedback effect. Consequently, such work facilitated byvarious embodiments also demonstrates how different of the level of NOand time affect to iNOS expression in cancer cells. Understanding theeffect of exogenous NO to apoptosis pathway can directly aid inspecifying improved carcinogenic therapy and efficient drug development.

Embodiments of the present application comprise an incubator system withchambers of the fluidic cell culture plates, which consists of anindividual gaseous regulator, thus allowing for the study of comparingthe cells responses to NO in normoxic and hypoxic condition at the sametime.

FIGS. 42b-c illustrate methods for comparing cell responses to NO innormoxic (low concentration and/or high concentration) and hypoxic (lowconcentration and/or high concentration), which can be conductedsimultaneously according to some embodiments. As illustrated in FIGS.42b and 42c , according to some embodiments, incubator system enablesthe test sample and control sample to be run simultaneously so that theexperimenter can observe different response for normoxia and hypoxia.

FIG. 42d illustrates a method for comparing cell responses to NO inhypoxic conditions, where a low concentration 0.5 μM porghryin stocksolution and a high concentration 2 μM porghryin stock solution areapplied, as well as various NO donor concentrations (10 nM, 100 nM, 1μM, 10 μM, 100 μM, and 1 mM) are applied.

Example Experiment Method Leveraging Various Embodiments

The main reagents are NO donor compounds, such as DEA/NO, DETA/NO,Sper/NO, PABA/NO, SNP, GTN, SNAP, and GSNO. An NO quantitative kit canbe used to detect the concentration of NO. Cancer cell lines can be, forexample, ovarian cancer cells such as OVCAR-3. The antibodies for suchan experiment can employ, for example, iNOS rabbit poly—clonal and IgG1: 1500. As described earlier, and MTT reagent can be used to analyzefor cellular proliferation, for example using a microtiter-plate reader.

Ovarian cancer cell lines, OVCAR-3 can be cultured in RPMI medium 1640(Life technologies, Gland Island, N.Y.) containing 10% FBS (HyClone) and1% of penicillin-streptomycin. A gas mixture comprising 94% nitrogen, 5%carbon dioxide, and 1% oxygen can be used to create a hypoxia condition;and a gas mixture comprising 95% air with 5% carbon dioxide can be usedto create a normoxia condition in humidified atmosphere. The cells canbe placed in well plates in Roswell Park Memorial Institute (RPMI)culturing media at a density of 1×104 cell/mL. The medium can be bubbledin advance with 100% N₂ before use so as to create a hypoxia condition.The cells can be then be cultured for an adequate duration to attainstability, for example for 48 hours, before use in the experimentalprocedures made possible by various embodiments.

In general, the cell culture requires mixing and washing steps; thus,the cells are transferred to a fume hood sterilely. The steps of mixingand washing induce a discontinued the growth condition which may affectto the results. For instance, the cells are cultured at 37° C. andhypoxia condition; but, since changing the media that needs to do in thefume hood, the temperature and the gaseous portion are changed. Though,minimizing the time of working outside the incubator may reduce theeffect of environmental changes on the cells' response, theenvironmental breaking of would be a major concern of human errors. Inaddition, the processes of washing and mixing may disturb a cellattachment; especially, the washing step has an effect on a cellpopulation. Therefore, if even feasible, such experimental proceduresrequires a skillful operator for reducing this error. However, thevarious incubator embodiments described earlier readily facilitate allaspects of these experimental procedures.

Various incubator embodiments described earlier provide selectivesegregated controlled support of cell culture environments andprocesses, all of which can be controlled in detail by computeralgorithm. Various incubator embodiments described earlier provideregulators that control the growth conditions such as temperature,humidity, gaseous portion and sterility as provided by other incubatorsavailable in the marketplace. However, various incubator embodimentsdescribed earlier additionally provide a plurality of controlledenvironments, and microfluidic systems allowing fluids such as cellculture media and other materials to be selectively transferred toindividually controlled cell culture wells without moving the cellculture microplates from the incubator. As a result, the growthcondition does not change during culturing, mixing and washing steps.Also, the mixing and washing processes are controlled by the computersystem, and the pressure of flowing solutions does not disturb the cellattachment. In these example ways, as well as many others, the variousincubator embodiments described earlier prevent vast numbers of problemsand susceptibilities to human errors, and can additionally significantlyreduce the amount of training required to perform experiments such asthis example as well as far more complex ones.

Measurement of the Concentration of Nitric Oxide for Nitrite Production

The releasing kinetic in hypoxic and normal conditions of various NOdonors such as DEA/NO, DETA/NO, Sper/NO, PABA/NO, SNP, GTN, SNAP, andGSNO can be studied by using NO quantitative kit as suggested by thetables depicted in FIG. 43a and FIG. 43b . Such example experiment-datacollection tables provide for capturing of cell exposure to differentconcentrations of NO from several kinds of NO donors at each time pointin normal condition and hypoxic conditions. After choosing the right NOdonor for testing each hypothesis, NO kit will be used for study ahalf-life and NO production at each concentration in hypoxic and normalenvironments. The results of this test will answer how much of NO donorand when NO donor will be added to the culture media to get the righttotal concentration of NO and maintain the steady state NO level,respectively.

FIG. 44 depicts an example representation of the effect of oxygenconcentration (pO₂) on O₂—, NO^(o) and ONOO— production [4],[34].Whereas O₂— is the dominant free radical species under normoxia, bothONOO— and O₂— are available under hypoxic conditions. Mitochondriallygenerated NO is expected to be the major mitochondrial free radicalspecies produced under anoxic conditions [25].

The study of Douglas D. Thomas et al. describe the method of NO and O2quantification. The steady—state concentration of NO was verified by twomethods; electrochemically and by a NO gas analyzer [22]. The signalswere calibrated by using argon—purged 100 mM phosphate buffer solutionsof saturated NO after measuring NO concentration (using 2,2′-azino-bis(ethylbenzothiazoline-6-sulfonic acid at 660 nm, 12,000 M⁻¹·cm⁻¹) [22].NO donors were exposed concurrently when culture media was changed tobetween hypoxia or normoxia conditions. Efimenko et al. observed theresponses of cells from exposing the cells to NO and other reactivespecies under hypoxia and nomoxia condition by using hypoxic incubator.The cell properties were analyzed immediately after cells had been takenoff the hypoxia incubator [23]. The NO concentration was determinedusing standard Griess reagent and the absorbance was measured at 560 nmusing a Tecan plate reader. These studies have demonstrated that theconcentrations of O2 are involved to a generating of O₂ ⁻, NO^(o) andONOO⁻. Under nomoxia, the majority of the reactive oxygen species is O₂⁻, however, under hypoxia, NO^(□) and ONOO⁻ are dominant [25].

Accordingly, an experiment that supports the hypothesis comprises theseinteracting factors requiring detailed consideration.

1. Concentration of O₂; hypoxia and normoxia,2. Concentrations of NO at steady state and other oxygen reactivespecies,3. Types of NO donors.,4. Time of exposure.

Concentration of O₂; Hypoxia and Normoxia

To study the cell responses to the gaseous environment, the cells areincubated in normoxia or hypoxia incubators and are takeninstantaneously for the cell analysis. For that reason, the errors fromcell transferring might occur; even though, a skillful person operatesthis procedure. In addition, due to a single chamber of incubator,comparing the responses of two samples—one for normoxia and another forhypoxia study-, the experiments are not be able to proceedconcomitantly; unless there are more than one incubator.

Various incubator embodiments described earlier provides microfluidicsystems within the microplate and monitoring features within themicroplate and incubator so that rapid careful transferring ofmicroplates out of the incubator and rapid evaluation or treatment isnot required. The cell culture environment can be selectively specifiedand constantly regulated to a desired condition—normia or hypoxia.Moreover, the incubator consists of multiple chambers of microfluidicplate and every chamber is sealed with a cap. Due to in fact thatseparate groups or even individual wells of cell culture can be provideda distinct gaseous condition, the samples exposing to normoxia andhypoxia can be experimental studied in a concurrent and immediatelycomparative fashion. For example, various incubator embodimentsdescribed earlier provide explicit support for the study of the cellresponses of different kinds of NO donor of both hypoxia and normoxiaenvironment occurs at the same time; in contrast, conventional cellincubators provide explicit barriers if not complete impediments to suchstudy and experiments.

Concentrations of NO at Steady State and Other Oxygen Reactive Species

In general, maintaining the level of the target drug in blood streamrequires the information of a half-life of the drug. The regimen of thedrug should be optimal, so it will help the concentration of the druglevel is in steady stead. For example, to maintain the concentration ofPhenobarbital, having a half-life as 100 hours, in steady state, theoral administration is repeat every 24 hr (for dosage of 100 mg), orevery 12 hr (for dosage of 50 mg). FIG. 45, adapted from [20]demonstrates the results of a simulation of Phenobarbital plasmaconcentration during repeated oral administration for the applicationschemes indicated. Based on Vd=38 L, CL=0.26 L/h (t1/2=100 h), F=1. FIG.46, adapted from [27], demonstrates steady state NO concentrationshowing NO generation from DETA/NO in the absence and presence ofanaerobically grown N. gonorrhoeae. Here DETA/NO was added to GCK to afinal concentration of 0.3 mM in the absence or presence (OD=0.025) ofN. gonorrhoeae strain F62 and the NO concentration was monitored asdescribed in the experimental procedures [26]. The study of Garthwaiteet al. could control the constant steady state concentration of NO overtime by balancing NO release from a NONOate, NO donor, with consumptionby a scavenger [28].

To study the effect of concentration of NO and time of cell exposure onthe cell responses, monitoring concentration of NO during cellincubation is required. In fact, a half-life of NO depends on NO donors.Also, NO donors have distinct behavioral NO releasing. Furthermore, NOproduction is relevant to the concentration of surrounding O₂ because NOis able to react with O₂ and create other oxygen reactive species.

Previous studies have developed NO delivery systems for controlledsteady state levels of NO and O₂ to imitate biological environments [29,30]. However, the measuring the concentration of NO during cell cultureis not convenient and can induce human errors because the cells neededto be transfer to an NO probe outside the incubator's environmentalcontrol, and as a result introduces significant risk of changing thebiological environments. In contrast, the various incubator embodimentsdescribed earlier provide the convenience of NO determination andminimizes the human errors and risks from changing the biologicalenvironment during short-term transferring of cell cultures outside ofthe incubator environment. Further, various incubator embodimentsdescribed earlier include biosensors provided within cell culture wellson microplates plates, making it possible to measure the levels of NO orother oxygen reactive species during cell incubation. Therefore, theincubated cells are continuously determined NO levels or other speciesin the environmental control incubator, and translocation the cellcultures outside the incubator is not required. In addition, variousincubator embodiments described earlier not only permits the control ofNO levels, but also can be used to prevent the production of otheroxygen reactive species by individual control of gas environments andmonitoring by sensors in the cell culture wells.

Types of NO Donors and Exposure Time

The cell responses depends on the concentration of NO and time of cellexposure. Additionally, each kind of NO donor has a different NOreleasing behavior. To compare the releasing behavior of each kind of NOdonor or the response of cells to NO concentration and time of NOexposure, multiple samples have to be measured concomitantly. Due to infact that comparing the samples may reduce the environmental errors,various incubator embodiments described earlier support the multipledeterminations leveraging multiple units of cell cultures (in the formof multiple separately controlled microplates, or multiple separatelygroups of wells within a microplate, or even multiple separatelyindividual wells), each containing individual gaseous control andsensors. Therefore, studying the effects of NO exposure parameters oncell responses by varying concentration of NO and time of cell exposureand the type of NO donors is advantageously enabled and supported viavarious incubator embodiments described earlier.

MTT Assay for Cellular Proliferation

To study the cellular proliferation of cancer cell lines, each type ofcancer cell line will be incubated with selective NO donor at theconcentration that will release steady optimal concentration. The celllines will be sampling at each time point as table 2 for studyingcellular proliferation by using MTT reagent. The inhibition rate (RT) ofthe selective NO donor can be calculated from the absorbance reading,which will be taken using a microtiter plate reader as stated by thefollowing formula:

% IR=(1−OD_(experiment)/OD_(control))×100

The results of this test will show when the culture cells will have thehighest % IR (what time point that NO will started inhibit cellularproliferation is) and what concentration will cause the maximum of % IR(the concentration of cell death). In addition, the results will presentthe range of the concentration of NO and time that cells can survive.

The study of Thomas observed the reduction of Alamar blue to itsfluorescence product (λ_(Ex/Em)=550/590 nm) for determining cellproliferation after the cell lines were seed into 96 well microtiterplates and treated in serum-free medium with NO donors for 24 hours[22]. Sato et al. quantified the number of early apoptotic and deadcells using flow cytometry with FITC—conjugated annexin V and propidiumiodide (PI). Annexin V-FITC- and PI-strained cells were excited by a 488nm laser light, and they were collected in the FITC (515-545 nm) and PI(600-620 nm) channels according to emission wavelength.

To determine cell proliferation, the previous findings have used thespectroscopic method, such as UV-Visible (MTT assay), and fluorescence[10, 22]. The methods also contain mixing and washing steps.Consequently, the sample is translocated from an incubator to a fumehood for mixing and washing, and to a spectroscopic microplate readerfor measuring an absorbance of an interested final product. As a result,the process of cell proliferative determination contains complex steps.According to the incubator embodiment, the microfluidic system, assignedto design the cell culture plate, is applied to resolve this problem.For example, the microfluidic system allows the MTT solution, from thestorage, react with mitochondria reducthase and produce the purplecrystals. Then the solvent flows from the solvent storage to dissolvethe purple crystals to be purple solution, which is observed by thespectroscopic apparatus implanted in the incubator. Therefore, thepresent application provides the convenience of automatic washing andmixing processes, and allows the reaction occur during incubationperiod.

Additional Example Embodiments of the Present Application

In one aspect, the present application provides the optimal biologicalenvironment such as temperature, gaseous portion, and humidification,wherein the apparatus comprises multiple-chamber controllers. Eachchamber provides individual environmental controlled system andindividual biosensor detection, and is operated independently.Furthermore, for the microplate comprises replaceable sensing optimizesthe cells for attaching and growing.

In certain embodiments, the incubator is able to read the signal fromthe chemical reaction or the cells responses by electrochemical andoptical biosensors within or attached to the microplate.

In certain embodiments, biosensors monitor chemical reactions duringcell culture, for instance measuring NO levels during cell incubation.

In certain embodiments, the microfluidic system allows solution oranalyte automatically flow through the target location; therefore, thewashing and mixing steps are controlled by a computer program.

In certain embodiments, the incubator comprises at least one selectivewavelength light source, which is beneficial for a certain experiment,such as the experiment that requires the particular wavelength for areaction.

In certain embodiments, the incubator comprises multiple chambers of theenvironmental control—each individual chamber has isolated temperatureand gas portion's controls, which allows cells to grow at differentcondition; and every chamber is able to operate concurrently andindependent of each other.

In certain embodiments, the incubator comprises multiple chambers ofisolated sensors. The isolated sensors consist a semiconductingmaterial, sensitive to chemical and biological changes. Moreover, theisolated sensors are available for studies of various concentrations,chemical reactions, and cell responses. As a result, every isolatedsensor is able to operate concomitantly.

In certain embodiments, the incubator comprises multiple chambers ofisolated light sources, which are beneficial for selective wavelengthchemical reactions.

In certain embodiments, a plurality of chemical compounds are selectedto degrade and release chemical moieties of interest at differentwavelengths, allowing a plurality of light sources to initiate ormodulate multiple reactions of an experiment at the same time.

In certain embodiments, the isolated sensors each comprise at least onelayer of a semiconducting material, wherein the semiconducting materialand the selective detection material form at least a portion of eachselective sensor, and wherein each selective sensor is configured toprovide a variation in an electrical signal responsive to the targetagent.

In certain embodiments, each of the isolated selective sensors isconnected to an electrical connection. In certain embodiments, theremovable medium apparatus further comprises an electrical interfacearrangement on the microplate, wherein the electrical interfacearrangement is electrically linked to the electrical connections of eachof the isolated electrical sensors and is further configured forelectrically linking to a host electrical interface within theincubator.

In certain embodiments, at least two of the selective sensors respond todifferent target agents in the analyte. In certain embodiments, at leasttwo of the selective sensors comprise a different selective detectionmaterial from each other, and wherein the different selective detectionmaterials respond to the same target agent in the analyte. In certainembodiments, the selective sensors are of the same nature.

In certain embodiments, at least two of the selective sensors are ofdifferent nature. In certain embodiments, the microplate comprises atleast one optical sensor and electrochemical sensor.

In certain embodiments, the microplate bottom allows optical propagationthrough it for a range of wavelengths usable by at least one opticalsensing arrangement.

In certain embodiments, the microplate bottom does not allow opticalpropagation through it for a range of wavelengths employed by at leastone optical sensing arrangement.

In certain embodiments, the microplate bottom further comprises anoptical filter.

In certain embodiments, the microplate bottom further comprises anoptical element. In certain embodiments, the fluid analyte is a raw orprocessed sample.

In certain embodiments, at least one of the selective sensors is anelectrochemical sensors, part of a field effecter transistor, or aphotodiode. In certain embodiments at least one of the selective sensormaterials comprises a molecularly imprinted material.

In certain embodiments, the molecularly imprinted material is amolecularly imprinted polymer. In certain embodiments, at least one ofthe selective sensor materials comprises an enzyme or a membrane.

In certain embodiments, the removable medium apparatus further comprisesa deposit of a reagent.

In certain embodiments, the reagent is a pH buffer material, or cellculture media, or other reagents.

In certain embodiments, the readable medium is attached to the substrateby printing at least one material on the substrate.

In certain embodiments, the readable medium is a separately manufacturedlabel that is adhered to the substrate.

In certain embodiments, the readable medium comprises one or more of:information usable to operate a testing procedure, information usable toperform a statistical analysis, data information, serial numberinformation, information specifying at least one algorithm, parametersused by at least one algorithm, optical encoded data, or magnetic strip.

In certain embodiments, the microplate further comprises a fluidicinterface arranged for providing fluid transfer for the receivingarrangement within the base unit.

In certain embodiments, the microplate is attached to a microplate capso that the resulting arrangement is configured to comprise a fluidchannel.

In certain embodiments, the microplate further comprises arrangementsassociated with at least one optical sensor.

In certain embodiments, the microplate cap further comprisesarrangements associated with at least one optical sensor.

In certain embodiments, the microplate provides a fluidic interfacearranged for providing fluid transfer for the receiving arrangementwithin the incubator.

In certain embodiments, the microplate allows optical propagationthrough it for a range of wavelengths usable by at least one opticalsensing arrangement.

In certain embodiments, the microplate cap does not allow opticalpropagation through it for a range of wavelengths employed by at leastone optical sensing arrangement.

In certain embodiments, the microplate cap further comprises an opticalfilter.

In certain embodiments, the microplate cap further comprises an opticalelement.

In certain embodiments, fluid routed through microfluidics in themicroplate comprises one or more of cells, viruses, suspensions,slurries, emulsions, micelles, or dissolved gases.

In certain embodiments, the incubator of the sensor device comprises atleast one computational processor for executing software and a receivingarrangement for receiving, aligning, or physically supporting theremovable medium apparatus.

In certain embodiments, the incubator further comprises an electricalinterface arrangement for electrically connecting to the removablemedium apparatus.

In certain embodiments, the incubator comprises interface electronicsfor connecting to the electrical interface arrangement for producingsensor measurement signals, each sensor measurement signal comprising ameasurement value, the measurement value being one from a range ofcollection of permitted values.

In certain embodiments, the incubator comprises a medium reader forreading encoded data on a readable medium on the removable mediumapparatus. In certain embodiments, the sensor device further comprises afluidic interface arrangement for connecting to the removable mediumapparatus.

In certain embodiments, the microplate comprises a fluid systemcomprising controllable valves that can be controlled by thecomputational processor and connected to the fluidic interfacearrangement.

Certain embodiments also provide a method of using any one of the sensordevices described herein for detecting a target agent in a fluidanalyte, comprising 1) allowing the fluid analyte to be in contact withthe selective detection material on the removable medium apparatus; and2) detecting a detectable signal from the selective sensor on theremovable medium apparatus, wherein a variation of the detectable signalprior to and after the contact of the fluid analyte is indicative of thepresence of the target analyte.

Also provided by the present application is a method of using any one ofthe sensor devices described herein for determining the amount of atarget agent in a fluid analyte, comprising: 1) allowing the fluidanalyte to be in contact with the selective detection material on theremovable medium apparatus, and 2) detecting a detectable signal fromthe selective sensors on the removable medium apparatus, wherein thechange of the detectable signal after the contact of the fluid analytecorrelates with the amount of the target agent in the fluid analyte.

In certain embodiments, the methods are used for detecting the cellresponses and monitor the concentration of the interested substancesduring cell culture.

In certain embodiments, the methods are used for a biological orchemical assay in the controlled environment of the incubator.

The present application also provides a method of making a microplatefor providing replaceable sensing function to an external base unit, theapparatus comprising a plurality of isolated selective sensors on thesurface of a substrate, wherein each of the isolated regions thesemiconducting material and selective detection material form at leastportions of a selective sensor, the method comprising: depositing anarray of isolated regions of semiconducting material on the surface of asubstrate, the isolated regions comprising at least one layer ofsemiconducting material; depositing at least one layer of a selectivedetection material on each of the isolated regions in the array.

In certain embodiments, the method further comprises providing anelectrical connection to each of the isolated regions of semiconductingmaterial.

In certain embodiments, the deposition is accomplished byinkjet-printing.

In certain embodiments, the deposition is accomplished by functionalprinting.

Closing

The terms “certain embodiments”, “an embodiment”, “embodiment”,“embodiments”, “the embodiment”, “the embodiments”, “one or moreembodiments”, “some embodiments”, and “one embodiment” mean one or more(but not all) embodiments unless expressly specified otherwise. Theterms “including”, “comprising”, “having” and variations thereof mean“including but not limited to”, unless expressly specified otherwise.The enumerated listing of items does not imply that any or all of theitems are mutually exclusive, unless expressly specified otherwise. Theterms “a”, “an” and “the” mean “one or more”, unless expressly specifiedotherwise.

The foregoing description, for purpose of explanation, has beendescribed with reference to specific embodiments. However, theillustrative discussions above are not intended to be exhaustive or tolimit the present application to the precise forms disclosed. Manymodifications and variations are possible in view of the aboveteachings. The embodiments were chosen and described in order to bestexplain the principles of the present application and its practicalapplications, to thereby enable others skilled in the art to bestutilize the present application and various embodiments with variousmodifications as are suited to the particular use contemplated.

While the present application has been described in detail withreference to disclosed embodiments, various modifications within thescope of the present application will be apparent to those of ordinaryskill in this technological field. It is to be appreciated that featuresdescribed with respect to one embodiment typically can be applied toother embodiments.

The present application can be embodied in other specific forms withoutdeparting from the spirit or essential characteristics thereof. Thepresent embodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the present applicationbeing indicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

Although exemplary embodiments have been provided in detail, variouschanges, substitutions and alternations could be made thereto withoutdeparting from spirit and scope of the disclosed subject matter asdefined by the appended claims. Variations described for the embodimentsmay be realized in any combination desirable for each particularapplication. Thus particular limitations and embodiment enhancementsdescribed herein, which may have particular advantages to a particularapplication, need not be used for all applications. Also, not alllimitations need be implemented in methods, systems, and apparatusesincluding one or more concepts described with relation to the providedembodiments. Therefore, the present application properly is to beconstrued with reference to the claims.

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What is claimed is:
 1. A cap system for use with a microplate comprisinga plurality of chamber-containing wells arranged in an array, the systemcomprising: a plurality of environment-localizing caps includingplurality of seals arranged in an array of environment-localizing caps,the array of environment-localizing caps arranged to align with thearray of wells of the microplate so that each of the caps separatelycover a top surface of a different associated well in the array of wellsand separately seals the top surface of the different associated well inthe array of wells, wherein each cap directly seals the entire topsurface of the different associated well and wherein each seal overlapsa portion of the top microplate, dedicated localized illuminationassociated with each of the individual wells wherein the array ofenvironment-localizing caps fitted with the array of wells create aseparate environment for each well.
 2. The cap system of claim 1,wherein the dedicated localized illumination is provided by at least oneLED.
 3. The cap system of claim 1, wherein each cap comprises at leastone LED.
 4. The cap system of claim 1, wherein each microplate well isilluminated by at least one dedicated localized LED.
 5. The cap systemof claim 1, wherein the illumination comprises visible rangewavelengths.
 6. The cap system of claim 1, wherein the illuminationcomprises ultraviolet (U.V.) range wavelengths.
 7. The cap system ofclaim 1, wherein the localized illumination provides a range ofillumination wavelengths.
 8. The cap system of claim 1, wherein thelocalized illumination provides a selectable range of illuminationwavelengths.
 9. The cap system of claim 1, wherein the localizedillumination provides a combination of illumination wavelengths.
 10. Thecap system of claim 1, wherein the localized illumination provides aselectable combination of illumination wavelengths.
 11. The cap systemof claim 1, wherein the localized illumination is configured to providelight for use in imaging.
 12. The cap system of claim 1, wherein thelocalized illumination is configured to provide light for opticallystimulation of a photosensitizer.
 13. The cap system of claim 1, whereinthe localized illumination is configured to provide light for opticallystimulation of a fluorescent probe.
 14. The cap system of claim 1,wherein the localized illumination is configured to provide light foroptically stimulation of a fluorescent marker.
 15. The cap system ofclaim 1, wherein the localized illumination is configured to providelight for optically stimulation of a fluorophore.
 16. The cap system ofclaim 1, wherein the localized illumination is configured to providelight for optically stimulation of a fluorescent chemosensor.
 17. Thecap system of claim 1, wherein the localized illumination is configuredto provide light for fluorescence imaging.
 18. The cap system of claim1, wherein a cap for an individual well further comprises at least onefluidic structure within the microplate to interface with the pluralityof wells and reagent deposits.
 19. The cap system of claim 1, wherein acap for an individual well further comprises at least one gas exchangestructure within the microplate to interface with the plurality of wellsand reagent deposits.
 20. The cap system of claim 1, wherein a cap foran individual well further comprises at least one sensor dedicated tothat individual well.
 21. The cap system of claim 3, wherein the LED isconfigured to emit light and as a wavelength-selective photo detector.